Therapeutic compounds

ABSTRACT

The invention provides novel JAK-3 inhibitors that are useful for treating leukemia and lymphoma. The compounds are also useful to treat or prevent skin cancer, as well as sunburn and UVB-induced skin inflammation. In addition, the compounds of the present invention prevent the immunosuppressive effects of UVB radiation, and are useful to treat or prevent autoimmune diseases, inflammation, and transplant rejection. The invention also provides pharmaceutical compositions comprising compounds of the invention, as well as therapeutic methods for their use.

[0001] DNA response elements in promoters of target genes (Demoulin, J.B., et al., Mol. Cell. Biol. 16: 4710-6, 1996). STATs then interactdirectly or indirectly, via their transactivation domain, withcomponents of the RNA polymerase II complex to activate transcription oftarget genes. Different ligands employ specific JAK and STAT familymembers, thus utilization of this pathway mandates specificity insignaling cascades and contributes to a diverse array of cellularresponses. Janus kinases, including JAK3, are abundantly expressed inprimary leukemic cells from children with acute lymphoblastic leukemia(ALL), the most common form of childhood cancer, and recent studies havecorrelated STAT activation in ALL cells with signals regulatingapoptosis (Demoulin, J. B., et al., Mol. Cell. Biol. 16: 4710-6, 1996;Jurlander, J., et al., Blood. 89: 4146-52, 1997; Kaneko, S., Suzuki, etal., Clin. Exp. Immun. 109: 185-193, 1997; and Nakamura, N., et al., J.Biol. Chem. 271: 19483-8, 1996).

[0002] Thus, JAK-3 is an important enzyme that plays an essential rolein the function of lymphocytes, macrophages, and mast cells. Compoundswhich inhibit JAK-3 would be expected to be useful for treating orpreventing diseases or conditions wherein the function of lymphocytes,macrophages, or mast cells is implicated, such as, leukemia, lymphoma,transplant rejection (e.g. pancreas islet transplant rejection, bonemarrow transplant applications (e.g. graft-versus-host disease),autoimmune diseases (e.g. diabetes), and inflammation (e.g. asthma,inflammation associated with sun burn, and skin cancer). A continuingneed exists for compounds and methods that are useful for the treatmentand/or prevention of such conditions and diseases.

SUMMARY OF THE INVENTION

[0003] The present invention provides JAK-3 inhibiting compounds thatare nontoxic in the administered dosage range. The JAK-3 inhibitors ofthe invention are useful for treating leukemia and lymphoma. Thecompounds are also useful to prevent skin cancer, as well as to treat orprevent sunburn and UVB-induced skin inflammation. In addition, thecompounds of the present invention prevent the immunosuppressive effectsof UVB radiation, and are useful to treat or prevent autoimmunediseases, inflammation, and transplant rejection.

[0004] The present invention provides a therapeutic method for treatingleukemia or lymphoma comprising administering to the mammal in needthereof an effective amount of a JAK-3 inhibitor.

[0005] The present invention also provides a therapeutic method forpreventing or reducing UV B radiation-induced inflammatory response in amammal comprising administering to the mammal in need thereof aneffective amount of a JAK-3 inhibitor.

[0006] The present invention also provides a therapeutic method forinhibiting the release of prostaglandin E₂ in a mammal comprisingadministering to the mammal in need thereof an effective amount of aJAK-3 inhibitor.

[0007] The present invention also provides a therapeutic method forpreventing or reducing UVB-induced skin edema or vascular permeabilitychanges in a mammal comprising administering to the mammal in needthereof an effective amount of a JAK-3 inhibitor.

[0008] The present invention also provides a therapeutic method forpreventing or reducing UV B radiation-induced damage to epithelial cellsor mutation frequency in skin in a mammal comprising administering tothe mammal in need thereof an effective amount of a JAK-3 inhibitor.

[0009] The present invention also provides a therapeutic method forprotecting a mammal from tumorigenic effects of UVB light comprisingadministering to the mammal in need thereof an effective amount of aJAK-3 inhibitor.

[0010] Representative JAK-3 inhibitors of the invention have also beenfound to exhibit significant anti-proliferative activity againstT-cells, and have been found to inhibit IL-2 dependent cellproliferation. Thus, the compounds can be used to treat or preventtransplant complications (e.g. rejection of a donor organ transplant bythe host immune system), and complications associated with bone marrowtransplantation such as graft versus host disease.

[0011] In addition, the compounds of the invention are effective intreating and preventing autoimmune diseases, such as insulin dependentdiabetes. The compounds are also effective in treating airwayinflammation (asthma).

[0012] Accordingly, the invention also provides a therapeutic method fortreating (or preventing) leukemia, transplant rejection, graft-verseshost disease, inflammation, asthma, autoimmune diseases includingdiabetes, and inflammation related cancer development in the skin,comprising administering to the mammal in need thereof an effectiveamount of a compound of formula I.

[0013] The invention also provides novel compounds of formula Idisclosed herein as well as pharmaceutical compositions comprisingcompounds of formula I.

[0014] A specific JAK-3 inhibitor useful in the medicamants and methodsof the invention is a compound of formula I:

[0015] wherein:

[0016] X is HN, R₁₁N, S, O, CH₂, or R₁₁CH;

[0017] R₁₁ is hydrogen, (C₁-C₄)alkyl, or (C₁-C₄)alkanoyl;

[0018] R₁-R₈ are each independently hydrogen, hydroxy, mercapto, amino,nitro, (C₁-C₄)alkyl, (C₁-C₄)alkoxy, (C₁-C₄)alkylthio, or halo; whereintwo adjacent groups of R₁-R₅ together with the phenyl ring to which theyare attached may optionally form a fused ring, for example forming anaphthyl or a tetrahydronaphthyl ring; and further wherein the ringformed by the two adjacent groups of R₁-R₅ may optionally be substitutedby 1, 2, 3, or 4 hydroxy, mercapto, amino, nitro, (C₁-C₄)alkyl,(C₁-C₄)alkoxy, (C₁-C₄)alkylthio, or halo; and R₉ and R₁₀ are eachindependently hydrogen, (C₁-C₄)alkyl, (C₁-C₄)alkoxy, halo, or(C₁-C₄)alkanoyl; or R₉ and R₁₀ together are methylenedioxy; or apharmaceutically acceptable salt thereof. Peferably, at least one of R₂and R₃ is hydroxy. More preferably, at least one of R₂ and R₃ ishydroxy, and one of R₁ to R₅ is halo.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1. Illustrates the synthesis of representative compounds offormula I from compound 5.

[0020]FIG. 2. [A] Model of JAK3 showing molecular surface of protein(blue), and catalytic (ATP binding) site (yellow). [B] Ribbonrepresentation (Cα backbone) of the homology model of the JAK3 kinasedomain. The WHI-P131 molecule is shown as a space-filling model in thecatalytic site of JAK3. [C] Close-up view of catalytic site of JAK3model with docked quinazoline inhibitor WHI-P131 (green). Residues andinhibitor are shown as space-filling atoms. The solvent-exposed openingof the catalytic site has dimensions to allow a relatively planarinhibitor to enter and bind to JAK3. The opening of the pocket isdefined by residues Pro906, Ser907, Gly908, Asp912, Arg953, Gly829,Leu828, and Tyr904 (blue residues). The far wall deep inside the pocketis lined with Leu905 (Cα backbone), Glu903, Met902, Lys905, and Asp967(pink residues), and the floor of the pocket is lined by Leu905 (sidechain), Val884, Leu956, and Ala966 (yellow residues). Residues definingthe roof of the pocket include Leu828, Gly829, Lys830, and Gly831(uppermost blue residues). Prepared using InsightII program.

[0021]FIG. 3. [A] Model of unoccupied space in the catalytic (ATPbinding) site of a JAK3 homology model. Shown in green is the bindingsite for ATP and the most likely binding site for dimethoxyquinazolineinhibitors. The green kinase active site region represents a totalvolume of approximately 530 Å. Modeling studies showed that an inhibitoror a portion of an inhibitor with significant binding to this regionwould occupy a volume less than 530 Å³ and have molecular dimensionscompatible with the shape of the binding site region. Other regions nearthe binding site which show measurable unoccupied volume are shown inroyal blue, pink, yellow, and light blue. These binding regions areeither unavailable to inhibitor molecules (royal blue) or representregions just large enough to occupy solvent molecules (pink, yellow,light blue). A model of WHI-P131 docked into the catalytic site is shownin white, superimposed on the green region. [B] Model of the catalyticsite of JAK3 with quinazolines WHI-P131 (multicolor), WHI-P132 (pink),and WHI-P154 (yellow). Each compound fits into the binding site butWHI-P132 (shown to be inactive against JAK3 in biological assays) lacksan OH group that is in a location to bind with Asp967. WHI-P131 andWHI-P154, with OH groups at the C4′ position of the phenyl ring, areable to form a favorable interaction with Asp967 of JAK3, which maycontribute to their enhanced inhibition activity. [C] Features ofdimethoxyquinazoline derivatives which are predicted to aid binding toJAK3 catalytic site.

[0022]FIG. 4. [A] Structural comparison of nonconserved residues in thecatalytic sites of 5 different protein tyrosine kinases: JAK3 (pink),BTK (red), SYK (light blue), IRK (dark blue), and HCK (yellow). Residueswithin 5 Å of the docked JAK3 inhibitor, WHI-P131 (white), are shown asrod-shaped side chains. The C alpha backbone of JAK3 is shown as a thinpink line, for perspective. Regions A to F correspond to areascontaining nonconserved residues in the catalytic site (see B andResults and Discussion). Crystal structure coordinates of HCK and IRK,and homology models of JAK3, BTK, and SYK were used for the structuralanalysis. [B] Nonconserved residues in the catalytic sites of 8different protein tyrosine kinases. Regions A-F refer to locations inthe catalytic site which are illustrated in A.

[0023]FIG. 5. Effects of WHI-P131 on the Tyrosine Kinase Activity ofJAK3. [A]-[D]. JAK3, JAK1, and JAK2 immunoprecipitated from Sf21 insectovary cells transfected with the appropriate baculovirus expressionvectors were treated with WHI-P131, then subjected to in vitro kinaseassays as described in Methods. The enzymatic activity of JAKs wasdetermined by measuring autophosphorylation in a 10 min kinase assay, asdescribed in Methods. The kinase activity (KA) levels were expressed aspercentage of baseline activity (%CON). [E] EMSAs of 32Dc22-IL-2Rβcells. WHI-P131 (10 μg/ml=33.6 μM) and WHI-P154 (10 μg/ml=26.6 μM) (butnot WHI-P132; 10 μg/ml=33.6 μM) inhibited IL-2 triggered JAK-3-dependentSTAT activation but not IL-3-triggered JAK-1/JAK-2-dependent STATactivation in 32Dc11-IL-2Rβ cells.

[0024]FIG. 6. Specificity of WHI-P131. JAK3, SYK, and BTKimmunoprecipitated from Sf21 insect ovary cells transfected with theappropriate baculovirus expression vectors, LYN immunoprecipitated fromNALM-6 human B-lineage ALL cells, and IRK immunoprecipitated from HepG2hepatoma cells were treated with WHI-P131, then subjected to in vitrokinase assays as described in Methods.

[0025]FIG. 7. WHI-P131 Depolarizes Mitochondrial Membranes in aConcentration-Dependent Fashion Without Affecting the MitochondrialMass. NALM-6 human leukemic cells were incubated with indicatedconcentrations of WHI-P131 for 48 h, stained with DiIC1 to assess themitochondrial membrane potential (Δψm) or NAO to detect themitochondrial mass and then analyzed with cell sorter equipped with HeNelaser. WHI-P131 caused a progressive increase in depolarizedmitochondria (as indicated by M1 in A) with increasing concentrations.At similar concentrations no significant change in mitochondrial mass[B] was detected. [C]: Cells were stained with JC-1 for simultaneousanalysis of mitochondrial mass (green fluorescence) and mitochondrialtransmembrane potential (red/orange fluorescence). Untreated NALM-6cells [D.1] as well as NALM-6 cells treated with 50 μM of WHI-P131 for24 hr [D.2] were incubated with JC-1 and analyzed by confocal laserscanning microscopy. Mitochondria of control cells showed a highermembrane potential (Δψm), as indicated by brighter JC-1 redfluorescence. Treatment of cells with WHI-P131 reduced mitochondrialmembrane Δψm as indicated by a substantial decrease in JC-1 redfluorescence.

[0026]FIG. 8. WHI-P131 Induces Apoptosis in NALM-6 Leukemia Cells. [A]:Cells were incubated with 10 μM-500 μM WHI-P131 for 24 hours and thenprocessed for in situ apoptosis assays. The percentage of apoptoticcells was determined by examining an average of 1380 cells/10fields/sample. Data points represent the mean from duplicate countsobtained in two independent experiments. [B.1], [B.2]: NALM-6 cells wereincubated with 100 μM of WHI-P131 for 48 hr, processed for the in situapoptosis assay and analyzed with a laser scanning confocal microscope.When compared with controls treated with DMSO (0.1%) [B.1], several ofthe cells incubated with WHI-P131 [B.2] showed apoptotic nuclei (yellowfluorescence). Red fluorescence represents nuclei stained with propidiumiodide.

[0027]FIG. 9. WHI-P131 Induces Apoptotic DNA Fragmentation in HumanLeukemia Cells. DNA from Triton-X-100 lysates of control and drugtreated cells was analyzed for fragmentation, as described (21). [A]NALM-6 human B-lineage ALL cells were treated for 24 hours at 37° C.with the listed dimethoxyquinazoline compounds at 1, 3, or 10 μM finalconcentrations. WHI-P131 was used as the lead JAK3 inhibitory compoundand all other compounds were included as controls which lacked JAK3inhibitory activity. [B] LC1;19 human B-lineage ALL cells and thecontrol cell lines SQ20B and M24-MET were treated for 24 hours at 37° C.with WHI-P131 at 1 or 3 μM final concentrations.

[0028]FIG. 10. Inhibitory effect of compound 6 on UVB-induced skinthickness in skh-1 mice. Female skh-1 mice were treated topically with 1mg/cm² of compound 6 prior to each UVB light exposure with 35 mj/cm².Skinfold thickness of each mouse was recorded twice weekly and theaverage of the two measurements was used in calculations. Data representmean±SEM (n=5-14). * P≦0.05 and ** P≦0.005 as compared to vehicletreated control.

[0029]FIG. 11. Inhibitory effect of compound 6 on the average number oflesions per mouse. Female skh-1 mice were treated topically with 1mg/cm² of compound 6 prior to each UVB light exposure with 35 mj/cm².The number of lesions was recorded twice weekly and the average of thetwo measurements was used in calculations. Data represent mean±SEM(n=5-14). * P<0.05 as compared to vehicle treated control.

[0030]FIG. 12. Inhibitory effect of Compound 6 on the average lesionvolume per mouse. Female skh-1 mice were treated topically with 1 mg/cm²of compound 6 prior to each UVB light exposure with 35 mj/cm². Thelesions greater than or equal to 1 mm in diameter were measured andrecorded twice weekly and an average of the two measurements was used incalculations. Lesion volume was calculated using the formula describedin Material and Methods. Data represent mean±SEM (n=5-14). * P≦0.05 ascompared to vehicle treated control.

[0031]FIG. 13. Morphological appearance of dorsal surface of mice after20 weeks of UVB irradiation. Female skh-1 mice were treated topicallywith 1 mg/cm² of compound 6 prior to each UVB light exposure with 35mj/cm. Mice were irradiated three times per week for a total of 20weeks. At 20 weeks the mice were anaesthetized, and a picture of theirdorsal surface was taken. Panel A, Unirradiated and vehicle treatedcontrol. Panel B, UVB irradiated and vehicle-treated mouse. Panel C, UVBirradiated and compound 6-treated mouse. Magnification 2×.

[0032]FIG. 14. Compound 6 inhibits UVB-induced PGE₂ synthesis. HaCaTcell cultures were either irradiated with UVB (25 mj/cm²) or shamirradiated. COMPOUND 6 (3-30(M) was added 60 min prior to irradiationand was readministered after UVB exposure. Cumulative PGE₂ releasedduring subsequent 6 h incubation was determined by EIA. Data representmean (SEM (n=3). FIG. 15. Compound 6 inhibits EGF-stimulated PGE₂release in epidermal cells. 50 ng/ml EGF was used to stimulate confluentHaCaT cell cultures both in the absence and presence of compound 6 andthe cells were incubated for 6 h at 37 (C. Following incubation thesupernatant was collected, and PGE₂ released during incubation wasdetermined by EIA. Data represent mean (SEM (n=5).

[0033]FIG. 16. Effect of Compound 6 on skinfold thickness of skh-1 micefollowing UVB light injury. Skh-1 mice were pretreated with compound 6(16 mg/kg; i.p. bolus injection) for 2 days. On the day of UVBirradiation the mice were anaesthetized and painted with 1.5 mg/cm²compound 6 on dorsal surface 15 minutes before UVB exposure, andirradiated with UVB light (250 mj/cm²). The skinfold thickness wasmeasured on day 1 through 5 post-irradiation. The data are expressed asmean (SEM (n=5-34).

[0034]FIG. 17. Inhibition of UVB-induced plasma exudation in skin ofskh-1 mice. Plasma exudation was evaluated at the times indicated afterUVB exposure (250 mj/cm²) by measuring the absorbence of Evans blue inskin extracts following the method described in Materials and Method.Data represent mean (SEM (n5-17).

[0035]FIG. 18. Effect of compound 6 on skin morphology at day 4 postUVB-irradiation. Skh-1 mice were pretreated with compound 6 (16 mg/kg;i.p. bolus injection) for 2 days, painted with 1.5 mg/cm² compound 6 ondorsal surface 15 minutes before UVB exposure, and irradiated with UVBlight (250 mj/cm²). On day 4 mice were anaesthetized and a picture oftheir dorsal surface was taken. Magnification 2×.

[0036]FIG. 19. Inhibition of UVB-induced histological changes in skin ofskh-1 mice by compound 6. Mice were treated with drug and exposed to UVB(250 mj/cm²) following the same procedure as described in Figure legend3. At 48 h post-irradiation mice were sacrificed, skin was biopsied andparaffin sections of the tissue were stained with hematoxylin and eosin.(a) control, (b) UVB (250 mj/cm²), and (c) COMPOUND 6+UVB (250 mj/cm²).Magnification 40×.

[0037]FIG. 20. Apoptosis of epidermal cells in UVB irradiated skin inskh-1 mice and its inhibition by compound 6. Skin biopsies from theirradiated and sham irradiated mice with or without drug treatment weretaken out at 48 h post UVB-irradiation and paraffin sections obtainedwere stained for apoptotic cells. The figure shows images captured byconfocal microscope using 60× magnification. Green stain in the pictureshows apoptotic cells.

[0038]FIG. 21. Dose-dependent supression of MLR (A), PHA-induced (B) andConA-induced (C) proliferation of splenocytes by WHI-P131. WHI-P131 wasadded in the concentration of 0.1, 1.0, 10, and 50 μg/mL during the5-day culture (MLR). or 3-day culture period (PHA and ConA).Proliferation was measured by WST-1 colorimetric assar. Results arepresented as mean O.D. ═SEM of 3-7 separate experiments. Statisticaldifferences between the groups analyzed by Student's t-test.

[0039]FIG. 22. Flow cytometry analysis of the percentage of apoptoticsplenocytes (TUNEL-positive) obtained after the 24-h-culture period withaddition of 0.1, 1, 10 and 100 μg/ml of WHI-P131. Results are presentedas mean ±SEM. Statistical differences between the groups analyzed byStudent's t-test.

[0040]FIG. 23. The in vivo prophylactic effect of WHI-P131administration on GVHD induced across the major histocompatibilitybarrier in C57BL/6 (H-2^(b)) recipients with BALB/c (H-2^(d))BM/splenocyte grafts. Irradiated (7.5 Gy) recipients were given BM andsplenocytes (25×10⁶ of each). Some recipients received syngeneic BM,while others were treated daily with 25 mg/kg of WHI-P131, 50 mg/kg ofWHI-P132, 3 mg/kg of Cyclosporine A, 10 mg/m² of Methotrexate or vehiclecontrol, as described in Material and Method section. Differences insurvival between the groups were analyzed by life-table analysis,Mantel-Cox test.

[0041]FIG. 24. The in vivo prophylactic effect of administration of drugcombinations presented in FIG. 23 on GVHD induced across the majorhistocompatibility barrier in C57BL/6 (H-2^(b)) recipients with BALB/c(H-2^(d)) BM/splenocyte grafts. WHI-P131 (25 mg/kg), cyclosporine A (3mg/kg) or combination of cyclosporine A and WHI-P131 (A); wereadministered i.p. Differences-in survival between the groups wereanalyzed by life-table analysis, Mantel-Cox test.

[0042]FIG. 25. The in vivo prophylactic effect of administration of drugcombinations presented in FIG. 23 on GVHD induced across the majorhistocompatibility barrier in C57BL/6 (H-2^(b)) recipients with BALB/c(H-2^(d)) BM/splenocyte grafts. WHI-P131 (25 mg/kg), and WHI-P131 andmethotrexate (10 mg/m²) or combination of methotrexate and WI-P131 wereadministered i.p. Differences in survival between the groups wereanalyzed by life-table analysis, Mantel-Cox test.

[0043]FIG. 26. shows the effects of compound 6 (60 mg/kg/day) on GVHDdevelopment.

[0044]FIG. 27. Cummulative diabetes incidence (A) inLDSTZ-treatedJak3-deficient and wild-type (WT) males studied during theexperimental period of 25 days post administration of first STZinjection; statistical difference obtained by life table analysis.

[0045]FIG. 28. Blood glucose level (mg/dl) (B) inLDSTZ-treatedJak3-deficient and wild-type (WT) males studied during theexperimental period of 25 days post administration of first STZinjection; statistical difference obtained by ANOVA.

[0046]FIG. 29. Cummulative diabetes incidence (A) in LDSTZ-treatedC57BL/6 males studied during the experimental period of 25 days postadministration of first STZ injection; statistical difference obtainedby life table analysis (Mantel-Cox test).

[0047]FIG. 30. Blood glucose level (mg/dl) in LDSTZ-treated C57BL/6males studied during the experimental period of 25 days postadministration of first STZ injection; statistical difference obtainedby ANOVA.

[0048]FIG. 31. Diabetes incidence in NOD females treated with 20 and 50mg/kg of WHI-P131 daily from 5 to 25 wk of age (A) statisticallysignificant differences between WHI-P131-treated and control mice wereobtained by life table analysis (Mantel-Cox test).

[0049]FIG. 32. Diabetes incidence in NOD females treated with 100 mg/kgof WHI-P131 from 5 or 10 to 25 wk of age; statistically significantdifferences between WHI-P131-treated and control mice were obtained bylife table analysis (Mantel-Cox test).

[0050]FIG. 33. IPGTT test performed in C57BL/6 females (20-wk-old),non-diabetic control NOD females (25-wk-old) and NOD females (25-wk-old)treated for 15 or 20 weeks with 100 mg/kg of WI-P131; statisticaldifferences obtained by Student's t-test: *, **, ***, **** p<0.05, 0.01,0.005 and 0.0001, respectively, compared to NOD vehicle-control group.

[0051]FIG. 34. Delayed adoptive transfer of diabetes into NOD-scidfemales by WHI-P131 treatment. NOD-scid females were transferred with10×10⁶ diabetic splenocytes and treated daily with 50 mg/kg of WHI-P131i.p. till diabetes onset. Statistically significant difference obtainedby life table analysis (Mantel-Cox test).

[0052]FIG. 35. Jak3^(−/−) mice do not reject islet allograft (A);hematoxilin and eosin (B) and immunostaining for insulin (C) ofallogeneic islet graft transplanted under the kidney capsule of diabeticJak3^(−/−) recipient sacrified on day 100 post transplantation. Datarepresentative of >30 sections/graft and 6 mice/group; ×10.

[0053]FIG. 36. WHI-P131 supresses MLR reaction. Responder splenocytes(4×10⁶/ml) were mixed with stimulator splenocytes (1.6×10⁶/ml) and drugswere added in the concentration of 0.1, 1, 10 and 50·g/ml during the5-day-culture period. Proliferation was measured by WST-1 colorimetricassay. Results are presented as mean O.D. ±SEM of 6 separateexperiments; p=0.0095 compared to proliferation of control cellsnot-exposed to the WHI-P131.

[0054]FIG. 37. Flow cytometry analysis of apoptotic splenocytes(TUNEL-positive). TUNEL staining was performed after 20 h incubationperiod of splenocytes (1.5×10⁶) with 0.1, 1, 10 and 100 μg/ml ofWHI-P131. Results are presented as mean ±SEM. Statistical differencesbetween the groups analyzed by Student's t-test.

[0055]FIG. 38. Effect of WHI-P131—(50 and 75 mg/kg), WHI-P132 (50 mg/kg)and cyclosporine A-treatment (20 mg/kg ) on islet allograft survival; pvalues obtained by life table analysis (Mantel-Cox test).

[0056]FIG. 39. Flow cytometry analysis of C57BL/6 splenocytes after thetreatment with 130 mg/kg of WHI-P131 for 10 days. Results are presentedas mean ±SEM. Statistical differences between the groups analyzed byStudent's t-test.

[0057]FIG. 40. Non-fasting blood glucose (mg/dl) in syngeneic islettransplant recipients treated with 50 mg/kg/day of WHI-P131 or vehiclecontrol during the period of 180 days post transplantation (A). Resultsare presented as mean ±SD.

[0058]FIG. 41. IPGTT of the same recipients in FIG. 39 andnon-transplanted control mice performed on day 70 (B) posttransplantation after the fasting period of 8 hours. Results arepresented as mean ±SEM (B, C).

[0059]FIG. 42. IPGTT of the same recipients in FIG. 39 andnon-transplanted control mice performed on day 180 (C) posttransplantation after the fasting period of 8 hours. Results arepresented as mean ±SEM.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The following definitions are used, unless otherwise described:halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, etc. denote bothstraight and branched groups; but reference to an individual radicalsuch as “propyl” embraces only the straight chain radical and referenceto an individual radical such as “isopropyl” embraces only the branchedchain radical. Aryl denotes a phenyl radical or an ortho-fused bicycliccarbocyclic radical having about nine to ten ring atoms in which atleast one ring is aromatic. Heteroaryl encompasses a radical attachedvia a ring carbon of a monocyclic aromatic ring containing five or sixring atoms consisting of carbon and one to four heteroatoms eachselected from the group consisting of non-peroxide oxygen, sulfur, andN(W) wherein W is absent or is H, O, (C₁-C₄)alkyl, phenyl or benzyl, aswell as a radical of an ortho-fused bicyclic heterocycle of about eightto ten ring atoms derived therefrom, particularly a benz-derivative orone derived by fusing a propylene, trimethylene, or tetramethylenediradical thereto.

[0061] Specific values listed below for radicals, substituents, andranges, are for illustration only; they do not exclude other definedvalues or other values within defined ranges for the radicals andsubstituents.

[0062] Specifically, (C₁-C₄)alkyl can be methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, or sec-butyl; (C₁-C₄)alkoxy can be methoxy,ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, or sec-butoxy;(C₁-C₄)alkylthio can be methylthio, ethylthio, propylthio,isopropylthio, butylthio, or isobutylthio; (C₁-C₄)alkanoyl can beacetyl, propanoyl, butanoyl, or isobutanoyl; aryl can be phenyl,indenyl, or naphthyl; and heteroaryl can be furyl, imidazolyl,triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl,pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide),thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or itsN-oxide) or quinolyl (or its N-oxide).

[0063] It will be appreciated by those skilled in the art that compoundsof the invention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine prostaglandin E₂ inhibitionactivity using the standard tests described herein, or using othersimilar tests which are well known in the art.

[0064] A specific group of compounds are compounds of formula I whereinX is R₁₁N. Another specific group of compounds are compounds of formulaI wherein X is HN.

[0065] A specific group of compounds are compounds of formula I whereinR₁, R₂, R₄, R₅, R₆, R₇, and R₁₀ are each H.

[0066] A specific group of compounds are compounds of formula I whereinR₃ is (C₁-C₄)alkoxy, hydroxy, nitro, halo, trifluoromethyl, or NR₁₂R₁₃wherein R₁₂ and R₁₃ are each independently hydrogen, (C₁-C₄)alkyl,(C₁-C₄)alkenyl, (C₁-C₄)alkynyl, (C₃-C₈)cycloalkyl, or heterocycle.Another specific group of compounds are compounds of formula I whereinR₃ is hydroxy.

[0067] A specific group of compounds are compounds of formula I whereinR₈ is (C₁-C₄)alkoxy. Another specific group of compounds are compoundsof formula I wherein R₈ is methoxy.

[0068] A specific group of compounds are compounds of formula I whereinR₉ is (C₁-C₄)alkoxy. Another specific group of compounds are compoundsof formula I wherein R₉ is methoxy.

[0069] A preferred compound is4-(4′-hydroxyl-phenyl)-amino-6,7-dimethoxyquinazoline WHI-P131; or apharmaceutically acceptable salt thereof.

[0070] A preferred compound is4-(3′-hydroxyl-phenyl)-amino-6,7-dimethoxyquinazoline WHI-P180; or apharmaceutically acceptable salt thereof.

[0071] A preferred compound is4-(3′,5′-dibromo-4′-hydroxyl-phenyl)-amino-6,7-dimethoxyquinazolineWHI-P97; or a pharmaceutically acceptable salt thereof.

[0072] A preferred compound is4-(3′bromo4′-hydroxyl-phenyl)-amino-6,7-dimethoxyquinazoline WHI-P154;or a pharmaceutically acceptable salt thereof.

[0073] The compounds of the present invention can be formulated aspharmaceutical compositions and administered to a mammalian host, suchas a human patient in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

[0074] Thus, the Substances may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the Substance may be combinedwith one or more excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparations should contain at least0.1% of the Substance. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 60% of the weight of a given unit dosage form. Theamount of Substance in such therapeutically useful compositions is suchthat an effective dosage level will be obtained.

[0075] The tablets, troches, pills, capsules, and the like may alsocontain the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, fructose, lactose or aspartame or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring may beadded. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active compound, sucrose or fructose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any unit dosage form should be pharmaceutically acceptableand substantially non-toxic in the amounts employed. In addition, theSubstance may be incorporated into sustained-release preparations anddevices.

[0076] The Substances may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the Substancecan be prepared in water, optionally mixed with a nontoxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, triacetin, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

[0077] The pharmaceutical dosage forms suitable for injection orinfusion can include sterile aqueous solutions or dispersions or sterilepowders comprising the Substance which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

[0078] Sterile injectable solutions are prepared by incorporating theSubstance in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

[0079] For topical administration, the Substances may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

[0080] Useful solid carriers include finely divided solids such as talc,clay, microcrystalline cellulose, silica, alumina and the like. Usefulliquid carriers include water, alcohols or glycols orwater-alcohol/glycol blends, in which the Substances can be dissolved ordispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied from absorbent pads, usedto impregnate bandages and other dressings, or sprayed onto the affectedarea using pump-type or aerosol sprayers.

[0081] Thickeners such as synthetic polymers, fatty acids, fatty acidsalts and esters, fatty alcohols, modified celluloses or modifiedmineral materials can also be employed with liquid carriers to formspreadable pastes, gels, ointments, soaps, and the like, for applicationdirectly to the skin of the user.

[0082] Examples of useful dermatological compositions which can be usedto deliver the Substances to the skin are known to the art; for example,see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No.4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S.Pat. No. 4,820,508).

[0083] Useful dosages of the compounds of formula I can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

[0084] Generally, the concentration of the Substance in a liquidcomposition, such as a lotion, will be from about 0.1-25 wt-%,preferably from about 0.5-10 wt-%. The concentration in a semi-solid orsolid composition such as a gel or a powder will be about 0. 1-5 wt-%,preferably about 0.5-2.5 wt-%.

[0085] The amount of the Substance required for use in treatment willvary not only with the particular salt selected but also with the routeof administration, the nature of the condition being treated and the ageand condition of the patient and will be ultimately at the discretion ofthe attendant physician or clinician.

[0086] In general, however, a suitable dose will be in the range of fromabout 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg ofbody weight per day, such as 3 to about 50 mg per kilogram body weightof the recipient per day, preferably in the range of 6 to 90 mg/kg/day,most preferably in the range of 15 to 60 mg/kg/day.

[0087] The Substance is conveniently administered in unit dosage form;for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

[0088] Ideally, the Substance should be administered to achieve peakplasma concentrations of from about 0.5 to about 75 μM, preferably,about 1 to 50 μM, most preferably, about 2 to about 30 μM. This may beachieved, for example, by the intravenous injection of a 0.05 to 5%solution of the Substance, optionally in saline, or orally administeredas a bolus containing about 1-100 mg of the Substance. Desirable bloodlevels may be maintained by continuous infusion to provide about0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15mg/kg of the Substance.

[0089] The Substance may conveniently be presented in a single dose oras divided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

[0090] The invention will now be illustrated by the followingnon-limiting Examples.

EXAMPLES

[0091] Melting points are uncorrected. ¹H NMR spectra were recordedusing a Varian Mercury 300 spectrometer in DMSO-d₆ or CDCl₃. Chemicalshifts are reported in parts per million (ppm) with tetramethylsilane(TMS) as an internal standard at zero ppm. Coupling constant (J) aregiven in hertz and the abbreviations s, d, t, q, and m refer to singlet,doublet, triplet, quartet and multiplet, respectively. Infrared spectrawere recorded on a Nicolet PROTEGE 460-IR spectrometer. Massspectroscopy data were recorded on a FINNIGAN MAT 95, VG 7070E-HF G.C.system with an HP 5973 Mass Selection Detector. UV spectra were recordedon BECKMAN DU 7400 and using MeOH as the solvent. TLC was performed on aprecoated silica gel plate (Silica Gel KGF; Whitman Inc). Silica gel(200-400 mesh, Whitman Inc.) was used for all column chromatographyseparations. All chemicals were reagent grade and were purchased fromAldrich Chemical Company (Milwaukee, Wis.) or Sigma Chemical Company(St. Louis, Mo.).

[0092] The synthesis of a representative compound of formula I isdescribed in Example 1. Other compounds of formula I can be preparedusing procedures similar to those described in Example 1.

Example 1 Chemical Synthesis and Characterization of4-(4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (6)

[0093]

[0094] 4,5-Dimethoxy-2-nitrobenzoic acid (1) was treated with thionylchloride and then reacted with ammonia to give4,5-dimethoxy-2-nitrobenzamide (2) as described by F. Nomoto et al.Chem. Pharm. Bull. 1990, 38, 1591-1595. The nitro group in compound (2)was reduced with sodium borohydride in the presence of copper sulfate(see C. L. Thomas Catalytic Processes and Proven Catalysts AcademicPress, New York (1970)) to give 4,5-dimethoxy-2-aminobenzamide (3) whichwas cyclized by refluxing with formic acid to give6,7-dimethoxyquinazoline-4(3H)-one (4). Compound (4) was refluxed withphosphorus oxytrichloride to provide compound (5), which is a usefulintermediate for preparing compounds of formula I wherein X is NH.Reaction of compound (5) with the requiste aniline provided thefollowing compounds of formula I:

Compound R₁ R₂ R₃ R₄ m.p. ° C. WHI-P97 H Br OH Br >300 WHI-P111 H Br MeH 225-228 WHI-P131 H H OH H 245-248 WHI-P132 OH H H H 255-258 WHI-P154 HBr OH H   233-233.5 WHI-P180 H OH H H 256-258 WHI-P197 H Cl OH H 245(dec) WHI-P292 OH H * * 277-279

[0095] Using a procedure similar to that described above, or usingprocedures which are known in the art for preparing other quinazolinecompounds, compounds of formula I can be prepared. For example,Compounds of formula I wherein X is S, O, or CH₂ can be prepared from acompound of formula (5) by reaction with a requsite compound of theformula PhXH as illustrated in FIG. 1.

Example 2 Identification of Selective JAK-3 Inhibitors

[0096] Constructing a Homology Model for the JAK3 Kinase Domain.

[0097] A homology model for JAK-3 was constructed as described by E. A.Sudbeck, et al., Clinical Cancer Research, 1999, 5, 1569-1582. Becausethe three dimensional coordinates of the JAK3 kinase domain arecurrently unknown, a structural model of JAK3 was required for a dockinganalysis of JAK3 inhibitors. A homology model of JAK3 was constructed(FIG. 2) by using known coordinates of homologous kinase domains as areference. The JAK3 homology model was built by first obtaining theprotein sequence of JAK3 (Swiss-Prot #P52333, Univ. of Geneva, Geneva,Switzerland) from GenBank (National Center for BiotechnologyInformation, Bethesda, Md.) and determining the most reasonable sequencealignment for the JAK3 kinase domain relative to some templatecoordinates (known kinase structures such as HCK, FGFR, and IRK(Sicheri, F., et al., Nature. 385: 602-9, 1997; Mohammadi, M., et al.,Cell. 86: 577-87, 1996; Mohammadi, M., et al., Science. 276: 955-60,1997; and Hubbard, S. R., et al., Nature. 372: 746-54, 1994). This wasaccomplished by first superimposing the Cα coordinates of the kinasedomains of HCK, FGFR, and IRK using the InsightII program to provide thebest overall structural comparison (InsightII, Molecular SimulationsInc. San Diego, Calif., 1996). The sequences were then aligned based onthe superimposition of their structures (amino acid sequences werealigned together if their Cα positions were spatially related to eachother). The alignment accommodated such features as loops in a proteinwhich differed from the other protein sequences. The structuralsuperimposition was performed using the Homology module of the InsightIIprogram and a Silicon Graphics INDIGO2 computer (Silicon Graphics,Mountain View, Calif.). The sequence alignment was done manually andproduced a sequence variation profile for each superimposed Cα position.The sequence variation profile served as a basis for the subsequentsequence alignment of the JAK3 kinase with the other three proteins. Inthis procedure, the sequence of JAK3 was incorporated into the programand aligned with the three known kinase proteins based on the sequencevariation profiles described previously. Next, a set of 3D coordinateswas assigned to the JAK3 kinase sequence using the 3D coordinates of HCKas a template and the Homology module within the InsightII program. Thecoordinates for a loop region where a sequence insertion occurs(relative to HCK without the loop) were chosen from a limited number ofpossibilities automatically generated by the computer program andmanually adjusted to a more ideal geometry using the program CHAIN(Sack, J. S., J. Mol. Graphics. 6: 244-245, 1988). Finally, theconstructed model of the JAK3 kinase domain was subjected to energyminimization using the X-PLOR program so that any steric strainintroduced during the model-building process could be relieved (Brünger,A. T. X-PLOR, A System for X-ray Crystallography and NMR). The model wasscreened for unfavorable steric contacts and if necessary such sidechains were remodeled either by using a rotamer library database or bymanually rotating the respective side chains. The procedure for homologymodel construction was repeated for JAK1 (SWISS-PROT #P23458) and JAK2(Genbank #AF005216) using the JAK3 model as a structural template. Theenergy minimized homology models of JAK1, JAK2, and JAK3 were then used,in conjunction with energy-minimized structural models ofdimethoxyquinazoline compounds, for modeling studies ofJAK/dimethoxyquinazoline complexes.

Docking Procedure Using Homology Model of JAK3

[0098] Kinase Domain.

[0099] Modeling of the JAK3/dimethoxyquinazoline complexes wasaccomplished using the Docking module within the program INSIGHTII andusing the Affinity suite of programs for automatically docking aninhibitor into a protein binding site (a similar procedure for EGFR andBTK was previously described, see Ghosh, S., et al., Clin. Can. Res. 4:2657-2668, 1998; Mahajan, S., et al., J. Biol. Chem. 274: 9587-9599,1999. The various docked positions of each compound were evaluated usinga Ludi (Bohm, H. J. et al., J. Comput. Aided Mol. Des. 8: 243-56, 1994)scoring procedure in INSIGHTII which estimated a binding constant,K_(i), taking into account the predicted lipophilic, hydrogen bonding,and van der Waals interactions between the inhibitor and the protein. Acomparison of the catalytic site residues of several different PTK wasmade by manually superimposing crystal structure coordinates of thekinase domains of IRK and HCK, and models of JAK1, JAK2, JAK3, BTK, andSYK and then identifying features in the active site which were uniqueto JAK3 (FIG. 3 and FIG. 4).

[0100] Chemical Synthesis of Quinazoline Derivatives.

[0101] The compounds listed in Table 1 were synthesized andcharacterized using literature procedures (Rewcastle, G. W., et al., J.Med. Chem. 38: 3482-7, 1995).

[0102] Table 1. Predicted Interaction of Protonated Quinazolines withJAK3 Kinase Active Site and Measured Inhibition Values (IC₅₀ values)from JAK3 Kinase Assays.

Mol. H Total Surf. Mol. Cmpd # H Bond Lipo Contact Binding Est. AreaVol. IC₅₀ Name R5′ R4′ R3′ R2′ Bonds Score Score Score Score Ki (μM)(Å²) (Å³) (μM) WHI-P131 H OH H H 3 188 476 64 568 2.3 276 261 9.1WHI-P97 Br OH Br H 3 156 559 65 622 0.6 314 307 11.0 WHI-P154 H OH Br H3 171 512 64 587 1.4 296 284 27.9 WHI-P79 H H Br H 1 9 531 63 444 36 278272 >300 WHI-P132 H H H OH 1 82 476 66 462 25 269 264 >300 WHI-P111 HCH₃ Br H 1 52 502 58 458 46 309 291 >300 WHI-P112 Br H H Br 0 0 541 62445 52 306 297 >300 WHI-P258 H H H H 0 0 510 64 414 72 266 252 >300

[0103] Immune Complex Kinase Assays.

[0104] Sf21 (IPLB-SF21-AE) cells (Vassilev, A., et al., J. Biol. Chem.274: 1646-1656, 1999) derived from the ovarian tissue of the fallarmyworm Spodotera frugiperda, were obtained from Invitrogen (Carlsbad,Calif.) and maintained at 26-28° C. in Grace's insect cell mediumsupplemented with 10% FBS and 1.0% antibiotic/antimycotic (GIBCO-BRL).Stock cells were maintained in suspension at 0.2-1.6×10⁶/ml in 600 mltotal culture volume in 1 L Bellco spinner flasks at 60-90 rpm. Cellviability was maintained at 95-100% as determined by trypan blue dyeexclusion. Sf21 cells were infected with a baculovirus expression vectorfor BTK, SYK, JAK1, JAK2, or JAK3. Cells were harvested, lysed (10 MMTris pH7.6, 100 mM NaCl, 1% Nonidet P40, 10% glycerol, 50 mM NaF, 100 μMNa₃VO₄, 50 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml aprotonin, 10μg/ml leupeptin), the kinases were immunoprecipitated from the lysates,and their enzymatic activity assayed, as reported (Vassilev, A., et al.,J. Biol. Chem. 274: 1646-1656, 1999; Uckun, F. M., et al., Science. 22:1096-1100, 1996; Goodman, P. A., et al., J. Biol. Chem. 273: 17742-48,1998; Mahajan, S., et al., Mol. Cell. Biol. 15: 5304-11, 1995; andUckun, F. M., et al., Science. 267: 886-91, 1995). Theimmunoprecipitates were subjected to Western blot analysis as previouslydescribed (Vassilev, A., et al., J. Biol. Chem. 274: 1646-1656, 1999;and Uckun, F. M., et al., Science. 22: 1096-1100, 1996).

[0105] For insulin receptor kinase (IRK) assays, HepG2 human hepatomacells grown to approximately 80% confluency were washed once withserum-free DMEM and starved for 3 hours at 37° in a CO₂ incubator.Subsequently, cells were stimulated with insulin (Eli Lilly and Co.,Indianapolis, Ind., cat# CP410;10 units/ml/10×10⁶ cells) for 10 minutesat room temperature. Following this IRK activation step, cells werewashed once with serum free medium, lysed in NP40 buffer and IRK wasimmunoprecipitated from the lysates with an anti-IRβ antibody (SantaCruz Biotechnology, Santa Cruz, Calif., Cat.# sc-711, polyclonal IgG).Prior to performing the immune complex kinase assays, the beads wereequilibrated with the kinase buffer (30 mM Hepes pH 7.4, 30 mM NaCl, 8mM MgCl₂, 4 mM MnCl₂). LYN was immunoprecipitated from whole celllysates of NALM-6 human leukemia cells as previously reported (Uckun, F.M., et al., Science. 267: 886-91, 1995; and Uckun, F. M., et al.,Journal of Biological Chemistry,. 271: 6396-6397, 1996).

[0106] In JAK3 immune complex kinase assays (17, 22), KL-2EBV-transformed human lymphoblastoid B cells (native JAK3 kinase assays)or insect ovary cells (recombinant JAK3 kinase assays) were lysed withNP-40 lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 5 mM EDTA, 1% NP-40,100 μM sodium orthovanadate, 100 μM sodium molybdate, 8 μg/ml aprotinin,5 μg/ml leupeptin, and 500 μM PMSF) and centrifuged 10 min at 13000× gto remove insoluble material. Samples were immunoprecipitated withantisera prepared against JAK3. The antisera were diluted and immunecomplexes collected by incubation with 15 μl protein A Sepharose. After4 washes with NP-40 lysis buffer, the protein A Sepharose beads werewashed once in kinase buffer (20 mM MOPS, pH7, 10 mM MgCl₂) andresuspended in the same buffer. Reactions were initiated by the additionof 25 μCi [γ³²P] ATP (5000 Ci/mMole) and unlabeled ATP to a finalconcentration of 5 μM. Reactions were terminated by boiling for 4 min inSDS sample buffer. Samples were run on 9.5% SDS polyacrylamide gels andlabeled proteins were detected by autoradiography. Followingelectrophoresis, kinase gels were dried onto Whatman 3M filter paper andsubjected to phosphorimaging on a Molecular Imager (Bio-Rad, Hercules,Calif.) as well as autoradiography on film. For each drug concentration,a kinase activity index (KA) was determined by comparing the kinaseactivity in phosphorimager units (PIU) to that of the baseline sample.In some experiments, cold kinase assays were performed, as described by(Uckun, F. M., et al., Clin. Can. Res. 4: 901-912, 1998).

[0107] Electrophoretic Mobility Shift Assays (EMSAs).

[0108] EMSAs were performed to examine the effects ofdimethoxyquinazoline compounds on cytokine-induced STAT activation in32Dc11/IL2Rβ cells (gift from Dr. James Ihle, St. Jude Children'sResearch Hospital), as previously described (Goodman, P. A., et al., J.Biol. Chem. 273: 17742-48, 1998).

[0109] Mitochondrial Membrane Potential Assessment.

[0110] To measure the changes in mitochondria, cells were incubated withWHI-P131 at concentrations ranging from 7.4 μg/ml (25 μM) to 30 μg/ml(200 μM) for 24 h or 48 h, stained with specific fluorescent dyes andanalyzed with flow cytometer. Mitochondrial membrane potential (Δψm) wasmeasured using two dyes including a lipophillic cation5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethlybenzimidazolylcarbocyanineiodide (JC-1) and a cyanine dye,1,1′,3,3,3′,3′-hexamethylindodicarbocyanine iodide (DiIC1 (27-29))obtained from Molecular Probes (Eugene, Oreg.). JC-1 is a monomer at 527nm after being excited at 490 nm; with polarization of Δψm, J-aggregatesare formed that shift emission to 590 nm (30). This can be detected on aflow cytometer by assessing the green signal (at 527 nm) andgreen-orange signal (at 590 nm) simultaneously, creating an index of thenumber of cells polarized and depolarized mitochondria. DiIC1, a cyaninedye which is amphioatheic and cationic, concentrates in energizedmitochondria and has been used in a variety of studies to measure themitochondrial membrane potential (Fujii, H., et al., Histochem J. 29:571-581, 1997; Mancini, M., et al., J. Cell Biol. 138: 449-469, 1997;and Petit, P. X., et al., J. Cell Biol. 130: 157-167, 1995). Cells werealso stained with DiIC1 at 40 nM concentration for 30 min in the dark asdescribed for JC-1. The cells were analyzed using a Vantage BectonDickinson (San Jose, Calif.) cell sorter equipped with HeNe laser withexcitation at 635 nm and the fluorescence was collected at 666 nm.

[0111] Mitochondrial Mass Determination.

[0112] Relative mitochondrial mass was measured by using BectonDickinson Calibur flow cytometry and the fluorescent stain10-n-nonyl-acridine orange (NAO), which binds the mitochondrialphospholipid cardiolipin, that has been extensively used to provide anindex of mitochondrial mass (Maftah, A., et al., Biochem. Biophys. Res.Commun. 164: 185-190, 1989).

[0113] Results and Discussion for Example 2

[0114] Homology Model of JAK3 Kinase Domain.

[0115] The three-dimensional coordinates of JAK3 used in theprotein/inhibitor modeling studies were constructed based on astructural alignment with the sequences of known crystal structures ofthe kinase domains of three protein tyrosine kinases (PTKs) (kinasedomains of HCK (9), FGFR (11), and IRK (37)), as detailed in Materialsand Methods. FIG. 2A and 1B show the homology model of the JAK3 kinasedomain, which is composed of an N-terminal lobe and a C-terminal lobewhich are linked by a hinge region near the catalytic (ATP-binding)site. The catalytic site is a pocket located in the central region ofthe kinase domain, which is defined by two β-sheets at the interfacebetween the N and C lobes. The opening to the catalytic site is solventaccessible and facilitates binding of ATP. Small molecule inhibitors canalso bind to the catalytic site which results in an attenuation of PTKactivity by inhibiting ATP binding. An analysis of the JAK3 modelrevealed specific features of the catalytic site which can be describedas a quadrilateral-shaped pocket (FIG. 2C). The opening of the pocket isdefined by residues Pro906, Ser907, Gly908, Asp912, Arg953, Gly829,Leu828, and Tyr904 (blue residues, FIG. 2C). The far wall deep insidethe pocket is lined with Leu905 (Cα backbone), Glu903, Met902, Lys905,and Asp967 (pink residues, FIG. 2C), and the floor of the pocket islined by Leu905 (side chain), Val884, Leu956, and Ala966 (yellowresidues, FIG. 2C). Residues defining the roof of the pocket includeLeu828, Gly829, Lys830, and Gly831 (uppermost blue residues, FIG. 2C).FIGS. 2C and 3A illustrate that the catalytic site of the JAK3 model hasapproximate dimensions of 8×11×20 Å and an available volume for bindingof approximately 530 Å³. According to the model, the solvent exposedopening to the binding region would allow inhibitors to enter and bindif the molecule contains some planarity.

[0116] While most of the catalytic site residues of the JAK3 kinasedomain were conserved relative to other PTKs, a few specific variationswere observed (FIG. 4). These differences include an alanine residue inBTK, IRK, and HCK/LYN (region A, FIG. 4A) which changes to Glu in SYKand Pro906 in JAK3. At region B, a tyrosine residue is conserved in JAK3(Tyr904), BTK, and LYN, but changes to Phe in HCK (which is the onlyapparent residue difference between HCK and LYN relevant to inhibitorbinding), Met in SYK, and Leu in IRK. Region C shows a methionineresidue which is conserved in BTK, IRK, and HCK/LYN, but changes toLeu905 in JAK3 and to Ala in SYK. Region D shows Met902 in JAK3, whichis conserved in SYK and IRK but changes to Thr in BTK and to a muchsmaller residue, Ala, in LYN and HCK. This Met902 residue in JAK3, whichis located on the back wall of the pocket and protrudes in toward thecenter of the pocket volume, can significantly affect the shape of thebinding pocket. At this location, the extended conformation of theMet902 side chain can hinder the close contact of inhibitors withresidues lining the back wall of the pocket and with the hinge region,relative to other kinases with smaller residues here such as BTK (Thr)and HCK/LYN (Ala). Ala966 in region E is conserved in HCK/LYN butchanges to Gly in IRK and to the more hydrophilic residue Ser in BTK andSYK. Region F, which is farther away from the inhibitor location, is theleast conserved region of the catalytic site and contains Asp912 inJAK3, Asn in BTK, Lys in SYK, Ser in IRK, and Asp in HCK/LYN (FIG. 4).These residue identity differences between tyrosine kinases provide thebasis for designing selective inhibitors of the JAK3 kinase domain.

[0117] Structure-based Design and Synthesis of JAK3 Inhibitors.

[0118] A computer docking procedure was used to predict how wellpotential inhibitors could fit into and bind to the catalytic site ofJAK3 and result in kinase inhibition (FIG. 3B). The dimethoxyquinazolinecompound WHI-P258 (4-(phenyl)-amino-6,7-dimethoxyquinazoline) containstwo methoxy groups on the quinazoline moiety but no other ringsubstituents. Molecular modeling studies using the homology model ofJAK3 kinase domain suggested that WHI-P258 would fit into the catalyticsite of JAK3, but probably would not bind very tightly due to limitedhydrogen bonding interactions. Asp967, a key residue in the catalyticsite of JAK3, can form a hydrogen bond with molecules binding to thecatalytic site, if such molecules contain a hydrogen bond donor groupsuch as an OH group. WHI-P258, however, does not contain an OH group andtherefore would not interact as favorably with Asp967. We postulatedthat the presence of an OH group at the 4′ position of the phenyl ringof WHI-P258 would result in stronger binding to JAK3 because of addedinteractions with Asp967. A series of dimethoxyquinazoline compoundswere designed and synthesized to test this hypothesis.

[0119] An estimation of the molecular volume for the compounds isprovided in Table 1.

[0120] A summary of structural features of the designeddimethoxyquinazoline compounds which were observed to be relevant forbinding to the catalytic site of JAK3 is shown in FIG. 3C. Theapproximate molecular volumes of the compounds in Table 1 range from 252Å to 307 Å, which are small enough to fit into the 530 Å³ binding siteof JAK3 kinase. Table 1 also lists the results of molecular modelingstudies including estimated binding constants (i.e., K_(i) values) forthe compounds which were docked into the JAK3 catalytic site. Thecompounds which were evaluated in docking studies contain substitutionsof similar functional groups at different positions on the phenyl ring.

[0121] The conformations of the energy-minimized docked models of thecompounds listed in Table 1 were relatively planar, with dihedral anglesof approximately 4-18° between the phenyl ring and quinazoline ringsystem. This conformation allows the molecule to fit more easily intothe catalytic site of JAK3. All of the listed compounds contain a ringnitrogen (N1), which can form a hydrogen bond with NH of Leu905 in thehinge region of JAK3. When N1 is protonated, the NH can instead interactwith the carbonyl group in Leu905 of JAK3. The presence of an OH groupat the 4′ position on the phenyl ring was anticipated to be particularlyimportant for binding to the catalytic site of JAK3. WHI-P131 (estimatedK_(i)=2.3 μM), WHI-P154 (estimated K_(i)=1.4 μM), and WHI-P97 (estimatedK_(i)=0.6 μM) shown in Table 1 were predicted to have favorable bindingto JAK3 and potent JAK3 inhibitory activity because they contain a 4′ OHgroup on the phenyl ring which can form a hydrogen bond with Asp967 ofJAK3, contributing to enhanced binding. However, the 2′ OH group ofWHI-P132 is not in the right orientation to interact with Asp967 and itprobably would form an intramolecular hydrogen bond with the quinazolinering nitrogen, which may contribute to a significantly lower affinity ofWHI-P132 for the catalytic site of JAK3. The relatively large brominesubstituents (WHI-P97, WHI-P154) can increase the molecular surface areain contact with binding site residues if the molecule can fit into thebinding site. Modeling of WHI-P154 and WHI-P97 showed that there isenough room to accommodate the bromine groups if the phenyl ring istilted slightly relative to the fused ring group of the molecule. Theresults from the modeling studies prompted the hypothesis that WHI-P131,WHI-P154, and WHI-P97 would exhibit potent JAK3-inhibitory activity. Inorder to test this hypothesis and validate the predictive value of thedescribed JAK3 homology model, we synthesized WHI-P131, WHI-P154,WHI-P97, and 5 other dimethoxyquinazoline compounds listed in Table 1.

[0122] Inhibition of JAK3 by Rationally Designed Dimethoxy-quinazolineCompounds.

[0123] We first used immune complex kinase assays to compare the effectsof the synthesized dimethoxyquinazoline compounds on the enzymaticactivity of human JAK3 immunoprecipitated from the KL2 EBV-transformedhuman lymphoblastoid B cell line. WHI-P131, WHI-P154, and WHI-P97, whichhad very similar estimated K_(i) values ranging from 0.6 μM to 2.3 μMand were predicted to show significant JAK3 inhibitory activity atmicromolar concentrations (which was not the case for the othercompounds which had estimated Ki values ranging from 25 μM to 72 μM),inhibited JAK3 in concentration-dependent fashion. The measured IC₅₀values were 9.1 μM for WHI-P131, 11.0 μM for WHI-P97, and 27.9 μM forWHI-P154, but >300 μM for all the other dimethoxyquinazoline compounds(Table 1). WHI-P131 and WHI-P154 were also tested against recombinantmurine JAK3 expressed in a baculovirus vector expression system andinhibited JAK3 in a concentration-dependent fashion with an IC₅₀ valueof 23.2 μg/ml (˜78 μM, FIG. 5A) and 48.1 μg/ml (˜128 μM, FIG. 5B),respectively. The ability of WHI-P131 and WHI-P154 to inhibitrecombinant JAK3 was confirmed in 4 independent experiments. Thesekinase assay results are consistent with our modeling studies describedabove.

[0124] Importantly, WHI-P131 and WHI-P154 did not exhibit any detectableinhibitory activity against recombinant JAK1 or JAK2 in immune complexkinase assays (FIGS. 5C and 5D). Electrophoretic Mobility Shift Assays(EMSAs) were also performed to confirm the JAK3 specificity of thesedimethoxyquinazoline compounds by examining their effects oncytokine-induced STAT activation in 32Dc11/IL2Rβ cells. As shown in FIG.5E, both WHI-P131 (10 μg/ml=33.6 μM) and WHI-P154 (10 μg/ml=26.6 μM)(but not the control compound WHI-P132, 10 μg/ml=33.6 μM) inhibitedJAK3-dependent STAT activation after stimulation with IL-2, but they didnot affect JAK1/JAK2-dependent STAT activation after stimulation withIL-3. Modeling studies suggest that this exquisite JAK3 specificitycould in part be due to an alanine residue (Ala966) which is present inthe catalytic site of JAK3 but changes to glycine in JAK1 and JAK2. Thisalanine group which is positioned near the phenyl ring of the bounddimethoxyquinazoline compounds can provide greater hydrophobic contactwith the phenyl group and thus can contribute to higher affinityrelative to the smaller glycine residue in this region of the bindingsite in JAK1 and JAK2.However, an accurate interpretation of theseremarkable differences in sensitivity of JAK3 versus JAK1 and JAK2 toWHI-131 and WHI-P154 will need to await the determination of the X-raycrystal structures of these kinases since simple amino aciddiscrepancies in their catalytic sites could result in pronouncedstructural differences.

[0125] Specificity of WHI-P131 as a Tyrosine Kinase Inhibitor.

[0126] Compound WHI-P131 was selected for further experiments designedto examine the sensitivity of non-Janus family protein tyrosine kinasesto this novel dimethoxyquinazoline class of JAK3 inhibitors. Theinhibitory activity of WHI-P131 against JAK3 was specific since it didnot affect the enzymatic activity of other protein tyrosine kinases(Table 1, FIG. 6), including the ZAP/SYK family tyrosine kinase SYK(FIG. 6C), TEC family tyrosine kinase BTK (FIG. 6D), SRC family tyrosinekinase LYN (FIG. 6E), and receptor family tyrosine kinase IRK (FIG. 6F)even at concentrations as high as 350 μM.

[0127] A structural analysis of these PTKs was performed using thecrystal structures of HCK (which served as a homology model for LYN) andIRK, and constructed homology models of JAK3, BTK, and SYK. Thisanalysis revealed some nonconserved residues located in the catalyticbinding site of the different tyrosine kinases which may contribute tothe specificity of WHI-P131 (FIG. 4). One such residue which is locatedclosest to the docked inhibitors is Ala966 in JAK3 (shown in region E inFIG. 4) which may provide the most favorable molecular surface contactwith the hydrophobic phenyl ring of WHI-P131. The fact that WHI-P131 didnot inhibit LYN, even though LYN contains the Ala residue conserved inJAK3 (Ala966), suggests that other factors (residue differences)contribute to this selectivity. Other nonconserved residues in thecatalytic site of tyrosine kinases are shown in regions A to F (FIG. 4).All of these differences in residues, especially residues which directlycontact the bound inhibitor, may play an important role in the observedspecificity of WHI-P131 for JAK3.

[0128] Example 2 demonstrates that a novel homology model of the JAK3kinase domain can be used for structure-based design and synthesis ofpotent and specific inhibitors of JAK3.

[0129] Finally, the homology model uniquely indicates that the activesite of JAK3 measures approximately 8 Å×11 Å×20 Å with an approximate530 Å³ volume available for inhibitor binding. Our modeling studiesusing the constructed homology model of JAK3 kinase also showed thatthere is significant opportunity for improvement of the quinazolineinhibitors. The JAK3 model shows that there is additional volume in theATP-binding site which can be better utilized by quinazolinederivatives. The average molecular volume of our dimethoxyquinazolinecompounds is 277 A³, which is well below the estimated total volume ofthe binding site, 530 A³. This leaves opportunities for the design ofnew inhibitors which have slightly larger functional groups at the 2′and 3′ positions of the phenyl ring. Structural and chemical features ofdimethoxyquinazoline compounds which are proposed to facilitate theirbinding to the Jak3 catalytic site include the following features whichare illustrated in FIG. 3C: 1) The presence of a 4′-OH group on thephenyl ring, 2) the presence of a hydrogen-bond acceptor (N, carbonyl,OH) near Leu905 NH, or a hydrogen-bond donor (NH, OH) near the Leu905carbonyl, 3) a relatively planar molecular shape to allow access to thebinding site, 4) the ability to fit into a 530 Å³ space defined by theresidues lining the Jak3 catalytic site These predicted bindingpreferences to JAK3 residues in the catalytic site can be used for thedesign of new and more potent inhibitors of JAK3.

[0130] The ability of a compound to act as an anti-leukemic agent can bedetermined using assays that are known in the art, or can be determinedusing assays similar to those described in Example 3.

Example 3 Leukemia Assays

[0131] The following cell lines were used in various biological assays:NALM-6 (pre-B-ALL), LC1;19 (Pre-B-ALL), DAUDI (B-ALL), RAMOS (B-ALL),MOLT-3 (T-ALL), HL60 (AML), BT-20 (breast cancer), M24-MET (melanoma),SQ20B (squamous cell carcinoma), and PC3 (prostate cancer). These celllines were maintained in culture as previously reported (16, 17, 20, 24,32, 33). Cells were seeded in 6-well tissue culture plates at a densityof 50×10⁴ cells/well in a treatment medium containing variousconcentrations of compound 6 and incubated for 24-48 hours at 37° C. ina humidified 5% CO₂ atmosphere.

[0132] To test the cytotoxicity of compound 6 against JAK-3 expressinghuman leukemia cells, leukemic cells were exposed to this JAK3 inhibitorand monitored for apoptosis-associated changes in mitochondrial membranepotential (Δψm) and mitochondrial mass using specific fluorescentmitochondrial probes and multiparameter flow cytometry. To measurechanges in Δψm, DiIC1 (which accumulates in energized mitochondria) wasused, whereas the mitochondrial mass was determined by staining thecells with NAO, a fluorescent dye that binds to the mitochondrial innermembrane independent of energetic state. Treatment of NALM-6 leukemiacells with compound 6 at 7.4 μg/ml (25 μM) to 60 μg/ml (200 μM) for 24 hor 48 h increased the number of depolarized mitochondria in aconcentration- and time-dependent manner as determined by flow cytometryusing DiIC1 (27-29) (FIG. 7A). As shown in FIG. 7A, the fraction ofDiIC1-negative cells with depolarized mitochondria increased from 1.3%in vehicle treated control cells to 81.6% in cells treated with 200 μMcompound 6 for 48 hours. The average EC₅₀ values for compound 6 induceddepolarization of mitochondria, as measured by decreased DiIC1 staining,were 79.3 μM for a 24 hour treatment and 58.4 μM for a 48 hourtreatment. The observed changes in Δψm were not due to loss inmitochondrial mass, as confirmed by a virtually identical stainingintensity of NAO in the treated and untreated NALM-6 cells (FIG. 7B). Tofurther confirm this relative change in Δψm, we used JC-1, amitochondrial dye, which normally exists in solution as a monomeremitting green fluorescence and assumes a dimeric configuration emittingred fluorescence in a reaction driven by mitochondrial transmembranepotential (Smiley, S. T., et al., Proc. Natl. Acad. Sci. U.S. A. 88:3671-3675, 1991). Thus, the use of JC-1 allows simultaneous analysis ofmitochondrial mass (green fluorescence) and mitochondrial transmembranepotential (red/orange fluorescence). After treatment of NALM-6 cellswith compound 6 at increasing concentrations ranging from 25 μM to 200μM and with increasing duration of exposure of 24 h or 48 h, we observeda progressive dissociation between Δψm and mitochondrial mass, withdecrement in JC-1 red/orange fluorescence without a significantcorresponding drop in JC-1 green fluorescence (FIG. 7C&D).

[0133] As shown in FIG. 7C, the fraction of JC-1 red/orangefluorescence-negative cells decreased from 79.2% in vehicle-treatedcontrol cells to 16.9% in cells treated with 200 μM compound 6 for 48hours. The corresponding values for JC-1 green fluorescence were 99.3%for vehicle-treated cells and 99.8% for compound 6-treated (200 μM×48hours) cells. The average EC₅₀ values for compound 6 induceddepolarization of mitochondria, as measured by decreased JC-1 red/orangefluorescence were 94.2 μM for a 24 hour treatment and 50.4 μM for a 48hour treatment. FIG. 7D compares the single color (red/orange)fluorescent confocal images of vehicle-treated and compound 6-treated(100 μM×48 hours) NALM-6 cells stained with JC-1. These resultscollectively demonstrate that compound 6 causes a significant decreasein mitochondrial transmembrane potential in NALM-6 human leukemia cells.Apoptosis Assays Cells were examined for apoptotic changes aftertreatment with compound 6 by the in situ terminal dideoxynucleotidyltransferase-mediated dUTP end-labeling (TUNEL) assay using the ApopTagapoptosis detection kit (Oncor, Gaithersburg, Md.) according to themanufacturer's recommendations, as detailed in our earlier reports (Zhu,D. -M., et al., Clin. Can. Res. 4: 2967-2976, 1998; D'Cruz, O., P., G.,and Uckun, F. M. Biology of Reproduction. 58: 1515-1526, 1998).

[0134] To detect apoptotic fragmentation of DNA, cells were harvestedafter a 24 hour exposure at 37° C. at 1, 3, and/or 10 μM concentrations.DNA was prepared from Triton-X-100 lysates for analysis of fragmentation(21). In brief, cells were lysed in hypotonic 10 mmol/L Tris-HCl (pH7.4), 1 mmol/L EDTA, 0.2% Triton-X-100 detergent; and subsequentlycentrifuged at 11,000 g. To detect apoptosis-associated DNAfragmentation, supernatants were electrophoresed on a 1.2% agarose gel,and the DNA fragments were visualized by ultraviolet light afterstaining with ethidium bromide.

[0135] To confirm that compound 6 can induce apoptosis in leukemiacells, the TdT-mediated labeling of 3′-OH termini withdigoxigenin-conjugated UTP using the in situ TUNEL assay method combinedwith confocal laser scanning microscopy was employed. At 48 hours aftertreatment with compound 6 at concentrations ranging from 10 μM to 500μM, NALM-6 cells were examined for digoxigenin-dUTP incorporation usingFITC-conjugated anti-digoxigenin (green fluorescence) and propidiumiodide counterstaining (red fluorescence). The percentage of apoptoticcells increased in a concentration-dependent fashion with an averageEC₅₀ value of 84.6 μM (FIG. 8A). FIG. 8B.1 and 8B.2 depict the two-colorconfocal microscopy images of vehicle-treated control cells and cellstreated with 100 μM compound 6. Compound 6-treated cells showedapoptotic yellow nuclei (=superimposed green and red fluorescence) (FIG.8B.2). Further evidence for apoptosis was observed in DNA fragmentationassays. Because of their exquisite sensitivity in detecting DNAfragments released from a small percentage of apoptotic cells, the DNAgel assays of apoptosis are uniquely suited to examine the nonspecifictoxicity of new antileukemic agents.

[0136]FIG. 9A demonstrates that supernatants from NALM-6 leukemia cells,treated with 1 μM or 3 μM COMPOUND 6, contained oligonucleosome-lengthDNA fragments with a “ladder-like” fragmentation pattern consistent withapoptosis, whereas no DNA fragments were detected in supernatants ofNALM-6 cells treated with structurally similar dimethoxyquinazolinecompounds which lacked JAK3 inhibitory activity. Unlike JAK3-positiveleukemia cells (NALM-6 cells in FIG. 9A and LC1;19 cells in FIG. 9B),JAK3 negative SQ20B squamous carcinoma cells and M24-MET melanoma cellsdid not show any evidence of apoptotic DNA fragmentation after treatmentwith compound 6 (FIG. 9B).

[0137] Taken together, these results provided experimental evidence thatthe JAK3 specific tyrosine kinase inhibitor compound 6 results indepolarization of the mitochondrial membrane and triggers apoptoticdeath in human B-lineage ALL cells, as evidenced by the ladder-likefragmentation pattern of nuclear DNA and digoxigenin-11-UTP labeling ofthe exposed 3′-hydroxyl end of the fragmented nuclear DNA in thepresence of TdT.

[0138] Clonogenic Assays

[0139] The antileukemic activity of compound 6 against clonogenic tumorcells was examined using a methylcellulose colony assay system (Uckun,F. M., et al., J. Exp. Med. 163: 347-368, 1986; Messinger, Y., et al.,Clin Cancer Res. 4: 165-70, 1998). In brief, cells (10⁵/ml in RPMI-10%FBS) were treated overnight at 37° C. with compound 6 at varyingconcentrations. After treatment, cells were washed twice, plated at 10⁴or 10⁵ cells/ml in RPMI-10% FMS-0.9% methylcellulose in Petri dishes,and cultured for 7 days at 37° C. in a humidified 5% CO₂ incubator.Subsequently, leukemic cell (or tumor cell) colonies were enumeratedusing an inverted phase-contrast microscope. The percent inhibition ofcolony formation was calculated using the following formula:

% Inhibition=1−mean number of colonies in test culture×100 mean numberof colonies in control culture

[0140] The antileukemic activity of compound 6 was measured bydetermining its ability to inhibit the in vitro clonogenic growth of theALL cell lines NALM-6, DAUDI, LC1;19, RAMOS, MOLT-3, and the AML cellline HL-60. As detailed in Table 1, compound 6 inhibited clonogenicgrowth in a concentration-dependent fashion with EC₅₀ values of 24.4 μMfor NALM-6 cells and 18.8 μM for DAUDI cells. At 100 μM, compound 6inhibited the in vitro colony formation by these leukemia cell linesby >99%. In contrast, compound 6 did not inhibit the clonogenic growthof JAK3-negative M24-MET melanoma or SQ20B squamous carcinoma cell linesTABLE 1 Effects of Compound 6 Against Clonogenic Leukemic Cells.Experiment (6) Number Concn. (μM) Cell Lines* NALM-6 (pre-B ALL) BT20(Breast Cancer) 1 Mean No. of % Mean No. of % Colonies/10⁵ CellsInhibition Colonies/10⁵ Cells Inhibition 0 2890 (2660, 3120) N.A. 2676(2712, 2640) N.A. 0.1 2970 (2756, 3184) 0 N.D. N.D. 1 3180 (3080, 3136)0 N.D. N.D. 10 1932 (1864, 2000) 33.2 3298 (2940, 3656) 0 100   2 (2,2) >99.9  2190 (1632, 2748) 18.2 DAUDI (B-ALL) M24-MET (Melanoma) 2 MeanNo. of % Mean No. of % Colonies/10⁵ Cells Inhibition Colonies/10⁵ CellsInhibition 0 3950 (3100, 4800) N.A. 177 (120, 235) N.A. 0.3 2030 (1700,2360) 48.6 312 (238, 386) 0 1 2136 (1568, 2704) 45.9 287 (157, 418) 0 31406 (988, 1824) 64.4 390 (280, 500) 0 10 1149 (1054, 1244) 70.9 301(249, 353) 0 30  29 (12, 46) 98.0 599 (534, 664) 0 RAMOS (B-ALL) SQ20B(Squamous Carcinoma) 3 Mean No. of % Mean No. of % Colonies/10⁵ CellsInhibition Colonies/10⁵ Cells Inhibition 0 1286 (1164, 1408) N.A. 754(452, 1056) N.A. 30   2 (0, 4) 99.8 838 (600, 1076) 0 MOLT-3 (T-ALL)HL-60 (AML) Mean No. of % Mean No. of % Colonies/10⁵ Cells InhibitionColonies/10⁵ Cells Inhibition 0 1322 (1252, 1392) N.A. 1854 (1648, 2060)N.A. 100   1 (1, 1) >99.9   1 (1,1) >99.9

[0141] In other studies, compound 6 was shown to inhibit JAK3, but notother protein tyrosine kinases, including JAK2, SYK, BTK, LYN, and IRK.ALL cells express JAK2. Similarly, the Src family PTK LYN, Zap/Sykfamily PTK SYK, and Tec family PTK BTK are expressed in ALL cells andaffect their adhesion, proliferation, and survival (Vassilev, A., etal., J. Biol. Chem. 274: 1646-1656, 1999; Uckun, F. M., et al., Science.267: 886-91, 1995; Kristupaitis, D., et al., J. BioL Chem. 273 (15):9119-9123, 1998, and Xiao, J., et al., J. Biol. Chem. 271: 7659-64,1996). IRK is the only member of the receptor PTK family that has beendetected in leukemic cells, especially pre-B ALL cells with a t(1;19)translocation (4143). Since compound 6 does not inhibit these tyrosinekinases, its ability to kill ALL cells cannot be attributed to anonspecific inhibition of JAK2, LYN, SYK, BTK, or IRK in these cells(Kaplan, G. C., et al., Biochem. Biophys. Res. Commun. 159(3): 1275-82,1989; Newman, J. D., et al., Int. J. Cancer. 50(3): 500-4, 1992;Bushkin, I. and Zick, Y., Biochem. Biophys. Res. Commun. 172(2): 676-82,1990).

[0142] The above shows that a representative JAK-3 inhibitor of formulaI (Compound 6) is a useful therapeutic agent for treating acutelymphoblastic leukemia, and demonstrates that compound 6 triggersapoptosis in leukemia cells. Thus, potent and specific inhibitors ofJAK3, such as dimethoxyquinazolines of formula I, are useful fortreating acute lymphoblastic leukemia, which is the most common form ofchildhood cancer.

[0143] The ability of a compound to prevent or treat skin cancer can bedetermined using assays that are known in the art, or can be determinedusing assays similar to those described in Example 4.

Example 4 Skin Cancer Assays

[0144] Female, 6-7 weeks old, hairless albino mice (skh-1) werepurchased from Charles River Laboratories (Wilmington, Mass.) and werehoused in a controlled environment (12-h light/12-h dark photo period,22±1° C., 60±10% relative humidity), which is fully accredited by theUSDA (United States Department of Agriculture). Animals were caged ingroups of five in a pathogen free environment in accordance with therules and regulations of U.S. Animal Welfare Act, and NationalInstitutes of Health (NIH). All mice were housed in microisolator cages(Lab Products, Inc., NJ) containing autoclaved bedding. The mice wereallowed free access to autoclaved pellet food and tap water throughoutthe experiments. Animal care and the experimental procedures werecarried out in agreement with institutional guidelines.

[0145] Buffered formalin phosphate (10%) was obtained from Fisherscientific (Springfield, N.J.). Dimethyl sulfoxide (DMSO) and Phosphatebuffered saline (PBS) were purchased from Sigma (St. Louis, Mo.).

[0146] Ultraviolet lamps (8-FSX24T12/HO/UVB) that emit lightpredominantly in the UVB range (280-320 nm) were obtained from NationalBiological Corporation, Twinsburg, Ohio. The irradiance of the UVB lampswas determined before each irradiation, using a UVB meter (model—500Cobtained from National Biological Corporation, Twinsburg, Ohio). Forexposure to UV light, three mice were placed in an open 28 cm×10 cmplastic box that was divided in three compartments (one mouse percompartment). The plastic box was placed in the center, under the bankof UVB light, and the mice were irradiated with 35 mj/cm² dose of UVB.The exposure time for a 35 mj/Cm 2 dose of UVB varied from 30-34 s. Thedistance between UVB lamps and the surface receiving irradiation was 20cm.

[0147] Tumorigenesis Protocol:

[0148] Mice were divided into three groups containing 5-14 mice pergroup as shown in Table 1. The mice were treated topically with a singleapplication of Compound 6 (1.0 mg/cm², dissolved in DMSO) over a 2 cm²area on the dorsal surface before each UVB exposure. After 15 min.following Compound 6 application the mice were irradiated with UVB (35mj/cm). UVB exposure of mice was performed three times a week for atotal of 20 weeks. Control mice were treated with vehicle prior to UVBlight exposure. TABLE 1 Experimental Design and Treatment Regimen UVBGroup No. of Mice Treatment (35 mj/cm²) A  5 Vehicle − B 10 Vehicle + C14 Compound 6 (1 mg/cm²) +

[0149] The skin thickness of mice, and papillary skin lesions greaterthan 1 mm in diameter were measured and recorded twice every week andthe average of the two measurements was used in the calculations. Skinthickness, lesion number and lesion diameter measurements were limitedonly to the 2 cm area on the dorsal surface of mice that was beingtreated either with compound 6 or vehicle. Lesion volume were calculatedusing the formula:

Volume=4/3πr³

[0150] At the end of the study, 5-19 lesions from each group wererandomly biopsied, and fixed in 10% buffered formalin. Formalin-fixedspecimens were embedded in paraffin blocks, sectioned at 4 μm thickness,and stained in haematoxylin-eosin. The pathological evaluation of skinsections was performed by a Certified Veterinary Pathologist who wasunaware of the identity of specimens.

[0151] Pathological Classification of Tumors:

[0152] The pathological classification of tumors was as follows: 1)Cutaneous papilloma: a tumor papillomatous growth of acanthoticepidermis without invasive growth of tumor cells into the dermis. Tumorcells do not appear atypical. 2) Actinic keratosis: a hyperplastic,orthokeratotic, mildly to moderately acanthotic epithelium. 3) Floridactinic keratosis: actinic keratosis with mild to moderately acanthoticridges extending into the superficial dermis, resembling superficiallyinvasive squamous cell carcinoma. 4) Keratoacanthoma: a papillary growthwith a central keratin filled crater surrounded by hyperplastic,acanthotic stratified squamous epithelium. The leading edge of the tumorpushes into the underlying dermis. 5) Squamous cell carcinoma (SCC): atumor with atypical cell nests invading into superficial and mid dermis.

[0153] Results for Example 4.

[0154] Sunburn is a UV induced inflammatory reaction that ischaracterized by cutaneous vasodilatation (erythema), and an increase invascular permeability with exudation of fluid (edema) in the affectedskin. The UVB-induced increase in plasma exudation can be detected as anincrease in skinfold thickness at 24 h following irradiation (Berg, R.J., et al., 1998, J Invest Dermatol 110:405-9). Exposure of mice to UVBlight (group B) induced an increase in skinfold thickness as compared tothe skin thickness of control group mice (FIG. 10). In the compound 6treated group (group C) there was an increase in skin thickness ascompared to the control (group A); however, the increase in skinthickness was significantly less as compared to UVB-irradiated andvehicle-treated group of mice indicating the anti-inflammatory effect ofcompound 6. Since the mice were irradiated chronically three times aweek, a sustained increase in skin edema was observed during the courseof the study in UVB-irradiated mice (groups B & C). However, at anygiven time point the increase in skin edema was partially inhibited bycompound 6 in the drug treated mice.

[0155] Chronic exposure of skin to UVB radiations primarily leads to thedevelopment of fine skin lesions that can grow both in number as well assize with further exposures to such radiations. As shown in FIG. 11, thechronic UVB exposure of mice induced the appearance of fine lesions inthe affected skin that first appeared around 10 weeks of irradiation inthe vehicle-treated and UVB-irradiated group of mice (group B). Thetotal number of skin lesions increased exponentially with increasingnumbers of UVB-exposure in the UVB-irradiated group of mice (group B).However, in COMPOUND 6-treated mice (group C), the onset of lesions wasdelayed; the first lesion was observed after 14 weeks ofUVB-irradiation. Although the average number of lesions per mouse inthis group also increased with increasing numbers of UVB exposures as ingroup B, at any given time point, the average number of lesions permouse was always less as compared to its untreated control (group B).

[0156] This data indicate that application of compound 6 prior to UVexposure (a) delays the onset of tumor from 10 weeks of UVB treatment(group B) to 14 weeks, and (b) it inhibits the total number of lesionsresulting from repeated UVB exposure.

[0157] Following the first appearance around 10-14 weeks-of UVBirradiation, the skin lesions increased both in total number as well asin size with increasing number of UVB exposures. In order to compare thesize of skin lesions, lesion diameter was converted to lesion volumeusing the formula described in Materials and Methods, and average lesionvolume per mouse was compared. FIG. 12 shows the effect of compound 6treatment on average lesion volume per mouse. It is observed from thefigure that the average lesion volume increased with time in both of theUVB-irradiated groups of mice (groups B & C) but compound 6 treatment ofmice inhibited the increase in lesion volume (group C, FIG. 12 and Table2). In addition, we also compared the average volume per lesion incompound 6-treated and -untreated groups of mice after 20 weeks ofirradiation. As observed before, with average skin lesion volume permouse, the average volume per lesion was also inhibited by compound 6treatment (Table 2). TABLE 2 Inhibitory effect of topical administrationof Compound 6 on UVB- induced tumorigenesis in skh-1 mice No. oflesions/ Skin lesion volume/ Avg. Vol/ Group No of mice mouse mouselesion A  5 0 0 0 B 10 4.2 ± 1.6 10.6 ± 4.3 2.5 ± 0.5 C 14 1.6 ± 0.4 3.2 ± 0.9* 1.9 ± 0.5

[0158] Morphological and Histopathological Data.

[0159] The morphological appearance of mouse skin after 20 weeks ofirradiation from all three groups of mice has been compared. The FIG. 13shows that repeated exposure with UVB light induced the growth of anumber of lesions on the affected skin (FIG. 13, Panel B) whereastreatment with compound 6 inhibited the growth of such lesions.

[0160] Histopathological findings of skin biopsies in mice after 20weeks of UVB irradiation are presented in Table 3. TABLE 3 Inhibitoryeffect of topical administration of Compound 6 on UVB- inducedkeratoacanthoma and squamous cell carcinomas in skh-1 mice PercentagePercentage of mice of mice Percentage Percentage of with with of micemice with SCC/ No of Actinic Keratoacan- with Florid actinic Group micekeratosis thomas Papilloma keratosis A 5 0 0 0 0 B 10 60 10 10 80 C 1453 0 7 57

[0161] A mild to moderate dermatitis was observed in all three groupsincluding the unirradiated group of mice (group A) which could beinduced by topical application of the vehicle (DMSO) three times a weekduring the course of the study. Actinic keratosis, which represents ahyperplastic and mild to moderately acanthotic epidermis, was present inmost of the biopsies from both of the UVB irradiated groups (groups B&C). A single lesion of keratoacanthoma was observed in theUVB-irradiated and vehicle treated group of mice (group B). No suchlesion was observed in any of the 14 mice in compound 6 treated group(group C). The occurrence of superficially invasive squamous cellcarcinoma (Florid actinic keratosis and a precursor to SCC) and SCC wasnoted in both of the UVB irradiated groups (Group B &C), but in COMPOUND6 treated mice (group C) it was inhibited by about 23% (Table 3).Similarly, the incidence of cutaneous papilloma was also partiallyinhibited by compound 6.

[0162] The results of Example 4 indicate that the topical application ofcompound 6 prior to UV irradiation protects skin from the harmfulconsequences of skin cancer in chronic exposure. Specifically, a topicalapplication of compound 6 on skin before UVB light exposure markedlyinhibited the formation of skin lesions, decreased tumor size andinhibited the development of tumors in skh-1 mice. Extensivedocumentation has validated the role of UVB radiations in skin tumorformation (Devary, Y., et al., 1992, Cell 71:1081-91; Ley, R. D., etal., 1989, Photochem Photobiol 50:1-5;Hall, E. J., et al., 1988, Am JClin Oncol 11:220-52; and Marks, R. 1995, Cancer 75:607-12).UVB-irradiation of skin cells triggers the release of increased amountsof arachidonic acid and its metabolites (Konger, R. L., et al., 1998,Biochim Biophys Acta 1401:221-34. (12-15). Prostaglandins, which are theoxygenation product of arachidonic acid, are produced abundantlyfollowing UVB irradiation of skin cells (Hawk, J. L. M., and J. A.Parrish. 1993. Responses of Normal Skin to Ultraviolet Radiations.Plenum Medical Book Publishers, New York; Hruza, L. L., and A. P.Pentland. 1993, J Invest Dermatol 100:35S-41S; Kang-Rotondo, et al.,1993, Am J Physiol 264:C396401; and Grewe, M., U. et al., 1993, J InvestDennatol 101:528-31) and have been implicated in various models fortumorigenesis (Vanderveen, E. E., et al., 1986, Arch Dermatol122:407-12; Cerutti, P. A., and B. F. Trump. 1991, Cancer Cells 3:1-7).Evidences also indicate that in addition to stimulating tumor growth,prostaglandins have a tendency to suppress hosts' immune surveillance(Plescia, O. J., et al., 1975, Proc Natl Acad Sci U S A 72:1848-51;Goodwin, J. S. 1984. Am J Med 77:7-15 and thus, assist in tumorpromotion. Elevated levels of PGE₂ have been observed in squamous andbasal cell carcinomas of skin and may be correlated with the increasedmetastatic activity and invasive behavior (Vanderveen, E. E., et al.,1986, Arch Dermatol 122:407-12; Klapan, I., V. et al., 1992, J CancerRes Clin Oncol 118:308-13 of these tumors. Various chemical compoundswhich inhibit the production of prostaglandins have been observed toinhibit the growth of tumors (Snyderman, C. H., et al., 1995, ArchOtolaryngol Head Neck Surg 121:1017-20;Hial, V., et al., 1976, Eur JPharmacol 37:367-76; Lynch, N. R., et al., 1978, Br J Cancer 38:503-12.

[0163] The ability of compound 6 to inhibit the UVB induced skintumorigenesis combined with our previous observations that it inhibitsacute skin inflammation indicate that compound 6 is a usefulchemopreventive agent against some forms of human cancers induced byenvironmental agents, such as ultraviolet light.

[0164] The ability of a compound to prevent or treat skin cancer can bedetermined using assays that are known in the art, or can be determinedusing assays similar to those described in Example 5.

Example 5 UVB-induced Skin Carcinogenesis Assays

[0165] Compound 6 is able to significantly inhibit the UVB light inducedinflammatory mediator release in epidermal cells and thus it preventsthe inflammatory responses of UVB exposed skin.

[0166] Female, 6-7 weeks old, hairless albino mice (skh-1) werepurchased from Charles River Laboratories (Wilmington, Mass.). Thetransgenic BigBlue mice carrying multiple copies of the BigBlue (LIZshuttle vector which contains substrate for detection of mutations invivo were obtained from Stratagene (La Jolla, Calif.). Mice were cagedin groups of five in a pathogen free environment in accordance with therules and regulations of U.S. Animal Welfare Act, and NationalInstitutes of Health (NIH). Animal care and the experimental procedureswere carried out in agreement with institutional guidelines.

[0167] HaCaT, which is a spontaneously transformed human epidermal cellline (16) was maintained in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% fetal bovine serum (Summit Biotech, Ft. Collins,Colo.).

[0168] A bank of 8-FSX24T12/HO/UVB lamps (National BiologicalCorporation, Twinsburg, Ohio) that emits light predominantly in the UVBrange, 280-320 nm was used to irradiate mice and cell cultures. Theirradiance of UVB lamps was always measured before irradiation using aUVB meter (model—500C obtained from National Biological Corporation,Twinsburg, Ohio). The dose of UVB light used to irradiate cultures was25 mj/cm². Anesthetized mice received UVB radiations to a 2.0 cm² areaon their dorsal surface. This area was painted either with the testcompound (1.5 mg/cm²) in drug receiving mice, or with vehicle in controlmice fifteen minutes before irradiation. The distance between UVB lampsand surface receiving irradiation was 20 cm. The final radiation dosereceived by skh-1 hairless mice was 250 mj/cm² which is approximatelyseven times higher than the minimal erythema dose (MED) (17) in thesemice. BigBlue mice were shaved on their back before irradiation and thenexposed to 400 mj/cm² UVB light either in the presence or absence ofcompound 6.

[0169] Prostaglandin E2 Assay.

[0170] Confluent HaCaT cells cultured in 24-well culture dishes werewashed three times with serum-free DMEM containing 1% bovine serumalbumin (BSA) (Cayman chemicals, Ann Arbor, Mich.) and incubated with3-30 μM compound compound 6 for 1 hour at 37° C. After incubation thecells were washed twice with PBS, and either exposed to 25 mj/cm² UVBlight or stimulated with 50 ng/ml rhEGF, and fed with serum-free DMEMcontaining 1% BSA. compound 6 was readministered and the cells wereincubated for 6 h at 37° C. At 6 h following stimulation the cellsupernatant was collected and PGE₂ released in cell supernatants wasmeasured by a competitive EIA using an acetylcholine esterase-PGE₂tracer and anti-PGE₂ antibody supplied with the ELISA kit (Caymanchemicals, Ann Arbor, Mich.). Cellular protein was determined using thePierce's BCA protein assay method (Smith, P. K., et al., 1985, AnalBiochem. 150 (1):76-85).

[0171] Compound 6 was initially dissolved in dimethyl sulfoxide (DMSO)(Sigma, St. Louis, Mo.) at a concentration of 10 mg/ml and diluted to 1mg/ml concentration with Phosphate buffer saline (PBS) before injection.The mice were treated daily with 16 mg/kg i.p. bolus injection ofcompound 6 from day -2 (i.e. 2 days prior to UVB exposure) until thetermination of the experiment. Drug receiving mice were also paintedwith 1.5 mg/cm² of Compound 6, 15 min before irradiation. The controlmice received vehicle alone.

[0172] One of the major arachidonic acid metabolite in Ultraviolet lightB-irradiated keratinocytes is prostaglandin E₂ which can be detected asearly as 6 h, peaks between 24-48 h following UVB exposure, and inducesedema and erythema in skin (Gilchrest, B. A., et al., 1981, J Am AcadDermatol. 5 (4):411-22; Konger R. L., et al., 1998, Biochim. et Biophys.Acta (1401):221-34; Woodward, D. F., et al., 1981, Agents Actions. 11(6-7):711-7; Snyder, D. S., and W. H. Eaglstein. 1974. Br J Dermatol. 90(1):91-93; Snyder, D. S., and W. Eaglstein. 1974. J Invest Dermatol.62:47-50; and Gupta, N., and L. Levy. 1973, Br J Pharmacol. 47(2):240-8). We determined the effect of compound Compound 6 on PGE₂release in UVB irradiated epidermal cells. The human epidermal cells,HaCaT, were exposed to UVB light and incubated both in the presence orabsence of Compound 6 and cumulative PGE₂ released in cell supernatantsduring 6 h incubation was determined. Analysis of PGE₂ release (FIG. 14)showed that an exposure of HaCaTs to 25 mj/cm² UVB induced about eleven(11.11 (4.23) fold increase in PGE₂ level at 6 h post irradiation ascompared to non-UVB irradiated control. The UVB light inducedprostaglandin release was inhibited by Compound 6 in a concentrationdependent manner and about 90% inhibition in prostaglandin release wasobserved at 30 μM dose of Compound 6. This data indicated that Compound6 is able to inhibit prostaglandin E₂ release in UVB light-stimulatedepidermal cells.

[0173] Primary human keratinocytes as well as HaCaTs expresssignificantly high number of functional EGF-receptors on their cellsurface ( ). Upon stimulation the EGF-receptors present on the epidermalcells participate in transmembrane signaling and induce the formation ofprostaglandins. To determine that the previously observed inhibition inprostaglandin release by Compound 6 was due to the inhibition of EGF-Ractivation, we studied the effect of Compound 6 on EGF-stimulatedprostaglandin formation in epidermal cells. Stimulation of the HaCaTcells with 50 ng/ml rhEGF for 6 h induced about 4 fold increase inprostaglandin release over unstimulated control, (FIG. 15) and theobserved increase in prostaglandin release was inhibited in the presenceof 30 μM Compound 6, indicating that (i) epidermal growth factorreceptors mediate prostaglandin formation in epidermal cells, and (ii)Compound 6 inhibits the prostaglandin release in UVB light stimulatedcells through the inhibition of EGF-R activation.

[0174] Morphology and Skin Edema Measurement.

[0175] Morphological appearance of the UVB irradiated skin was monitoredvisually and compared with the control mice skin. Skinfold thickness ofthe dorsal surface of the mice was recorded before, and everyday afterUVB exposure for five days with a digital thickness gauge (Mitutoyo, So.Plainfield, N.J.) which measures thickness in 0-10 mm range with anaccuracy of 0.015 mm.

[0176] As mentioned earlier, Prostaglandin E₂ is a potent inflammatorymediator and is well known to induce vasodilation and potentate edema inskin following injury. Since our in vitro study using epidermal cellsshowed that compound Compound 6 inhibits the release of PGE₂ in UVBstimulated cells, we were interested to determine if Compound 6 is ableto inhibit the harmful inflammatory responses of skin following UVBlight exposure in vivo. For these studies we used female, hairless,albino skh-1 mice and exposed their dorsal surface with 250 mj/cm² UVBlight. The skinfold thickness of the dorsal surface of the mice wasdetermined as a measurement of skin edema from day 1 through day 5following irradiation. FIG. 16 shows that a single exposure of mice to250 mj/cm² UVB induced a time dependent increase in skinfold thickness.Compound 6 was not able to effectively inhibit the skin edema at 24 hpost irradiation. However, it blocked further increase in skin thicknessby 48 h post-UVB exposure in irradiated group of mice. In contrast, theskin thickness increased to about 70% of the control mice skin thicknessat the same time point. At 72 h post irradiation, a significant decreasein skin edema was observed in Compound 6 treated mice and by day fivepost irradiation, the skinfold thickness in drug treated mice was almostback to control level. In contrast, in vehicle treated group the skinthickness increased to a total of 2.5 fold of control mice skinthickness over five day period. Similar results were obtained with P131dissolved in polyethylene glycol-200 (PEG-200) (data not shown). Theseobservations indicated that Compound 6 is able to inhibit UVBlight-induced skin edema in mice.

[0177] We also monitored the morphological changes in skin appearancefollowing UVB light exposure in Compound 6 treated and vehicle treatedgroups of mice. Sunburn damage to the skin in first 24-48 h followingUVB exposure was visible as an “elephant skin” appearance of the skinsurface. The UVB light irradiated skin of mice appeared pink in color,leathery and thick. On day 1 and day 2 following the induction ofinflammation, no significant difference was noted in skin appearance ofdrug treated and vehicle treated groups of mice. However, in UVBirradiated, vehicle treated mice, starting at day 3 many flakes ofdesquamating skin could be seen peeling off the skin surface and by day5 the skin of this group of mice had become tough, leathery, and haddeveloped scars on the surface. In contrast, in Compound 6 treatedgroup, the skin appearance of mice improved following day 3 and by day 5post-UVB, the signs of skin inflammation had diminished and the skinappearance resembled to that of control group mice (FIG. 18).

[0178] UVB light-induced histological changes of the skin were alsostudied. The normal epidermis typically has a 2-3 cell layer andcontains scattered inflammatory cells especially around hair follicles(FIG. 19A). The UVB-irradiated skin showed thickened epidermis with 3-5cell layers. Large number of neutrophils were also accumulated in thedermis (FIG. 19B). In contrast, the skin of mice treated with Compound 6looked very much like the skin of unirradiated controls (FIG. 19C), with1-2 cell layers of epidermis and normal dermis. Thus, Compound 6prevented development of edema and neutrophil influx in UVB irradiatedskin of mice.

[0179] Vascular Permeability.

[0180] Vascular permeability was quantitatively assayed by leakage fromvessels of an albumin bound anionic dye, Evans blue (Sigma, St. Louis,Mo.). Evans blue (1%, 200 (1/mouse) was injected via the tail vein, 4 hlater the mice were killed and the dorsal irradiated skin was biopsied.From the biopsies the dye was extracted in formamide (Sigma, St. Louis,Mo.) by warming the samples at 80° C. for 2 h and the optical absorbanceof the formamide was measured at 620 nm.

[0181] Since edema is associated with increased plasma exudation, wedetermined the effect of Compound 6 on UVB induced skin vascularpermeability. The data on vascular permeability changes following theUVB irradiation has been presented in FIG. 17. Consistent with the skinedema findings there was an increase in vascular permeability of theskin in UVB irradiated mice. The effect of Compound 6 was minimal at 24and 48 h post irradiation. However, at day 5 post-UVB, in Compound 6treated group the vascular permeability was back to control levelwhereas a five fold increase in vascular permeability was observed inUVB exposed, vehicle treated group of mice.

[0182] Sun Burn Cell Staining and Histological Studies.

[0183] After the mice were killed by cervical dislocation, the skin wasremoved and spread on a sheet of dental wax. One punch (8 mm) was takenand fixed in buffered formalin. 4-5 μm thick sections were cut fromparaffin blocks. Sunburn cell staining was done using ApopTag Plus Insitu Detection kit (Oncor, Gaithersburg, Md.) which detects sunburncells by direct fluorescence of digoxigenin-labelled genomic DNA.Briefly, the residues of digoxigenin-nucleotides were catalyticallyadded to 3′-OH ends of double or single stranded DNA in presence ofterminal deoxynucleotidyl transferase enzyme and the bound nucleotideswere detected using anti-digoxigenin antibody conjugated withfluorescein.

[0184] To study the histological changes following irradiation thetissue sections were stained with hematoxylin and eosin and the stainedslides were examined microscopically.

[0185] An acute exposure to UVB light induces sunburn in skin cells. Thepresence of sunburn cells in UVB-irradiated mice skin was detected usingIn Situ apoptosis detection kit as stated earlier. A significant numberof sunburnt cells (FIG. 20) were observed in the skin of UVB-irradiatedmice at 48 h after light exposure. In contrast, in Compound 6 treatedmice significantly less or no sunburnt cells were observed at the sametime point. Thus, the data suggest that Compound 6 inhibits the UVBinduced cell death in mouse skin.

[0186] The ability of a compound to prevent or treat transplantcomplications can be determined using assays that are known in the art,or can be determined using assays similar to those described in Example6.

Example 6 Transplant Complications

[0187] Proliferation Assays.

[0188] Splenocytes (4×10⁵/100 μl) from 9-wk-old C57BL/6 males were usedas responders in phytohemagglutinin (PHA)- and concanavalin A (ConA)-induced proliferation assays. The cells were applied in triplicatesper group to a 96-well microplate in a final volume of 200 μl of RPMI1640 medium, supplemented by 10% fetal calf serum. PHA or Con A (Sigma,St. Louis, Mo.) were added in the concentration of 5 or 2 μg/ml,respectively. Compound 6 was added in the concentration of 0.1, 1, 10and 50 μg/ml. Cells were cultured in 5% CO₂ with humidified air in anincubator at 37° C. for 3 days. Then, a colorimetric assay for thequantification of cell proliferation, based on the cleavage of thetetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells(Boehringer Mannheim, Indianapolis, Ind.), was performed followingmanufacturer's instructions. The absorbance was measured at 450/690 nmon a Multiskan MS microplate scanner. The value of cell proliferationwas obtained by diminishing O.D. value of PHA- or Con A-stimulated cellproliferation by O.D. value of non-stimulated cells (negative control).

[0189] Mixed Lymphocyte Reaction (MLR).

[0190] For MLR assay, responder cells (splenocytes obtained from9-wk-old C57BL/6 males) were plated in triplicates in 96-well-plates inthe concentration of 4×10⁵/100 μl and mitomycin-treated stimulators(splenocytes obtained from 10-12-wk-old BALB/c males) were added in theconcentration of 8×10⁴ in 50 μl. Compound 6 was added in theconcentrations descried above to a final volume of 200 μl. Cells werecultured for 5 days and a colorimetric WST-1 assay was performed, asdescribed above.

[0191] Apoptosis Detection.

[0192] C57BL/6 splenocytes (3×10⁶/ml) were cultured in 24-well plate for24 h in 500 μl of RPMI 1640 under the conditions described above.Compound 6 was added in the concentrations of 0.1, 1, 10 and 100 μg/ml.Apoptotic cell death was detected by TUNEL, using InSitu Cell DetectionKit, Fluorescein (Boehringer Mannheim, Indianapolis, Ind.). After theculture period, cells were washed, fixed, permeabilised and stainedfollowing manufacturer's instructions and apoptosis was analyzed by flowcytometry, using FACS Calibur (Becton Dickinson, San Jose, Calif.).

[0193] Mice.

[0194] Bone marrow transplant recipients were 8-10 week old C57BL/6(H-2^(b)) male mice and donors were 6-8 week old BALB/c (H-2^(d)) males(both strains purchased at Taconic, Germantown, N.Y.). Mice were kept inAnimal care facility at The Hughes Institute, under thespecific-pathogen-free condition (SPF). Free access to standard mousediet (Harlan Teklad LM-485) and water was allowed. Recipients were givenantibiotic-supplemented water (sulfamethoxazole/trimethoprim, Hi-TechPharmacal, Amityville, N.Y.) starting the day before transplantation.

[0195] Irradiation.

[0196] Recipient mice, positioned in a pie shaped Lucite holder, weretreated one day prior to bone marrow transplantation with a lethal dose(7.5 Gy) of Cesium (JL Sheppard Labs, 47.08 rad/min).

[0197] Bone Marrow Transplantation (BMT).

[0198] Donor BALB/c bone marrow was collected into RPMI 1640 withL-glutamine (Cellgro) (Mediatech, Hendon, Va.) by flushing the shafts ofthe femur and tibia. At the same time, donor single cell suspension ofsplenocytes, eliminated from red blood cells by lysis buffer (ACK lysisbuffer -0.15M NH₄Cl, 1.0M KHCO₃, 0.01MNa₂EDTA) was prepared, as well. BMcells were suspended by agitation with a pasteur pipette and separatedfrom debris by passing through a fine pore nylon cell strainer. Redblood cells were eliminated by lysis buffer and clumps of debris wereallowed to settle out. The cells were washed and were resuspended fori.v. injection via the caudal vein. The standard inoculum consisted of25×10⁶ BM cells and 25×10⁶ splenocytes in 0.5 ml of RPMI 1640).

[0199] Graft-versus-host Disease (GVHD) Monitoring.

[0200] BMT recipeints were monitored daily for the onset of clinicalevidence of GVHD (determined by weight loss, manifestations of skinerythema, allopecia, hunching, diarrhea) and survival during the 90-dayobservation period. Survival times were measured from the day of BMT(day 0). Deaths occurring within 11 days of transplantation wereconsidered to be radiation-indiuced and were excluded.

[0201] Drug Treatments.

[0202] For GVHD prophylaxis—daily intraperitoneal (i.p.) injections ofCompound 6 (WM-P131),4-(3′-hydroxyl-phenyl)-amino-6,7-dimethoxyquinazoline (WHI-P132,Compound 7), Cyclosporine (Sandimmune® Sandoz Pharmaceuticals Ltd,Basle, Switzerland), Methylprednisolone (Depo-Medrol® Pharmacia & UpjohnCompany, Kalamazoo, Mich.), Methotrexate (Immunex Corporation, Seattle,Washington) and vehicle control were administered to mice starting theday before BMT (−1) or on the day of BMT (day 0). Compound 6 wasadministered in a dose of 25 mg/kg/day and 60 mg/kg/day (divided inthree doses), Compound 7-50 mg/kg/day day (divided in three doses), fromthe day 0 of BMT; cyclosporine, methylprednisolone and methotrexate wereinjected in a doses according the Children cancer group (CCG) protocol:3 mg/kg/day (divided in two doses), 10 mg/kg/day (divided in two doses)and 10 mg/m²/day (once daily), respectively. The treatments withcyclosporine and methylprednisolone started the day before BMT (−1) andlasted till the end of experimental period, while methotrexate wasadministered on days 1, 3, 6 and 11 post BMT.

[0203] Statistical Analysis.

[0204] Statistical analysis of data obtained in proliferation assays,MLR and apoptosis data was done by Student's t-test, while survival datawere analyzed by life-table methods using the Mantel-Cox test.

[0205] Data from the above assays is presented in FIGS. 21-26.

[0206]FIG. 21. Dose-dependent suppression of MLR (A), PHA-induced (B)and ConA-induced (C) proliferation of splenocytes by WHI-P131. WHI-P131was added in the concentration of 0.1, 1, 10 and 50 μg/ml during the5-day-culture (MLR) or 3-day-culture period (PHA and ConA assays).Proliferation was measured by WST-1 colorimetric assay. Results arepresented as mean O.D. ±SEM of 3-7 separate experiments. Statisticaldifferences between the groups analyzed by Student's t-test.

[0207] The ability of a compound to prevent or treat autoimmune diseases(e.g. autoimmune induced diabetes) be determined using assays that areknown in the art, or can be determined using assays similar to thosedescribed in Example 7.

Example 7. Autoimmune Diseases

[0208] C57BL/6 and NOD mice were purchased from Taconic, Germantown,N.Y., and housed under pathogen-free conditions in Animal care facilityof Hughes Institute. Jak3^(−/−) (C57BIJ6×129/Sv) mice, homologous fordisrupted Jak3 gene were generous gift of Dr. J. N. Ihle, St. JudeChildren's Research Hospital, Memphis, Tenn. A homozygous Jak3^(−/−)mice were bred to C57BL/6 mice and the offspring of the F1 generationwere backcrossed to C57BL/6 mice. After three generations ofbackcrossing to C57BL/6, the offspring were intercrossed to produceJak3^(−/−) and wild-type (WT)Jak3^(+/+) mice, which were used in ourexperiments.

[0209] Induction of LDSTZ Model of Diabetes.

[0210] C57BL/6 male mice or JAK3-deficient and WT mice (8-10-wk-old)were injected intraperitoneally with low-dose (40 mg/kg ) of STZ (Sigma,St Louis, Mo.) daily for 5 consecutive days, for induction of autoimmuneexperimental diabetes (LDSTZ). STZ was dissolved in citrate buffer pH4.0 on ice, and injected within 10 min of preparation. Mice weremonitored for diabetes development by testing blood glucose from thesecond week (day 7) after the STZ administrations by One Touch Profilediabetes tracking system (Lifescan, Milpitas, Calif.). Mice withglycemia over 220 mg/dl on three consecutive tests were considereddiabetic, with the first detection of chronic glycemia taken as the dateof diabetes onset. A group of C57BL/6 males was treated withWHI-P131-100 mg/kg/day, i.p., devided in two equal doses, from abeggining of the experiment till day 25. As WHI-P131 was solubilazed in10% DMSO in PBS, control mice were treated with 10% DMSO in PBS, usingthe same conditions as described above.

[0211] Treatment of NOD Females with WHI-P131 and Assessment of DiabetesDevelopment.

[0212] NOD females were treated from 5- or 10-wk of age with differentdose of WHI-P131, daily, i.p. Control mice were treated with 10% DMSO inPBS. Mice were monitored for diabetes development by testing bloodglucose from 10 wk of age by One Touch Profile diabetes tracking system,as described above.

[0213] Intraperitoneal Glucose Tolerance Test (IPGTT).

[0214] Intraperitoneal glucose tolerance test (IPGTI) was performed inthe group of non-diabetic WHI-P131-treated and vehicle-treated controlNOD females on the end of experimental period (25 wk of age of NODmice). Mice were fasted for 10 hours, and the glucose solution (1.5 g/kgbody weight) was injected i.p. Before and after injection of glucose,blood samples were taken and blood glucose levels were measured (asdescribed above) at 0, 30, 60 and 120 min time points.

[0215] Histology.

[0216] The group of control and WHI-P131-treated non-diabetic NODfemales was sacrificed at the end of experimental period, at 25 wksofage, and characteristic histopathologic lesion of islets (insulitis) wasevaluated in each mouse scoring at least 25 islets per mouse. Briefly,pancreas was removed, fixed in 10% formalin, parafin embedded, cut andstained with hematoxylin and eosin for light microscopic examinations.All islets sampled from three nonoverlapping pancreatic levels wereassigned an insulitis score as follows: 0—no visible lesions;1—peri-insulitis with no islet penetration; 2—<25% of the isletinfiltrated; 3—>25% of the islet infiltrated; 4—end stage.

[0217] Adoptive Transfer of Diabetic Splenocytes to NOD-scid/scidFemales and Assesment of Diabetes Development.

[0218] Single-cell suspensions of splenic leucocytes pooled from 8-10diabetic NOD females were prepared by passage through Nitex 110 mesh,and red blood cells were lysed in 10× Gey's solution. Aliquots of 1×10⁷splenocytes were adoptively transferred intravenously into 4-week-oldNOD/Lt-scid/scid females (The Jackson Laboratory, Bar Harbor, Me.).WHI-P131 treatment (50 mg/kg) or control treatment (10% DMSO in PBS) ofNOD-scid mice started at the same time. Mice were monitored for diabetesdevelopment by testing urinary glucose every week from second week afterthe transfer by Chemstrip uGK strips (Boehringer Mannheim, Indianapolis,Ind.). Mice with glycosuria over 500 mg/dl (+++) on consecutive weeklytests were considered diabetic, with the first detection of chronicglycosuria taken as the date of IDDM onset.

[0219] Statistical Analysis.

[0220] Statistical analysis was done by using unpaired Student's t-test(IPGTT and insulitis data) and ANOVA test (differences in glycemic levelbetween the experimental groups). Experimental differences in IDDMincidence studies in WHI-P131-treated and control NOD and LDSTZ-treatedmice or in adoptively transferred scid mice were assessed by theKaplan-Meier life table analysis using Mantel-Cox test. The p value<0.05 was considered as statistically significant.

[0221] Results for Example 7

[0222] Development of LDSTZ Diabetes is Inhibited in JAK3-deficient Mice

[0223]FIG. 27 shows cummulative diabetes incidence (A) and blood glucoselevel (B) in JAK3-deficient and WT mice treated by low-dose STZ. While13/13 (100%) of WT mice developed hyperglycemia till day 14 post firstSIZ injection, only {fraction (1/12)} (8.3%) of JAK3-deficient micebecame diabetic in entire experimental period of 25 days (p<0.0001)(FIG. 27). WT mice exhibited increase of blood glucose from day 7,reaching the hyperglycemic level (>220 mg/dl) on day 9 post first STZinjection (FIG. 28). In contrary, JAK3-deficient mice stayednormoglycemic throught entire experimental period (p<0.0001 compared toWT glycemic level by ANOVA) (FIG. 28).

[0224] Inhibition of Diabetes Development in LDSTZ Model of Disease byJAK3 Kinase Inhibitor WHI-P131.

[0225] Daily treatment of C57BL/6 males by 100 mg/kg of WHI-P131 (2doses) i.p., initiated from the first day of SIZ injections, inhibitedthe development of LDSTZ diabetes (FIG. 29). While {fraction (20/38)}(52.6%) of control mice developed diabetes on day 7, followed by{fraction (32/38)} (84.2%) of diabetic mice on day 9 and {fraction(36/38)} (97.4%) of diabetic mice on day 11 post first STZ injection,only {fraction (4/20)} (20%) WHI-P131-treated mice became diabetic onday 7, followed by {fraction (10/20)} (50%) on day 9 and {fraction(13/20)} (65%) on day 11 (FIG. 29). FIG. 30 shows that WHI-P131-treatedmice exhibited significantly lower. (p=0.027) blood glucose levelthrought entire experimental period compared to the control mice.

[0226] Prevention of IDDM Development in NOD Females by WHI-P131Treatment.

[0227] The diabetes incidence in NOD females treated daily with: a) 20and 50 mg/kg of WHI-P131 from 5 to 25 wks of age (FIG. 27), and b) 100mg/kg of WHI-P131 from 5 to 8, 5 to 25 and 10-25 wks of age (FIG. 28)was studied. While diabetes appeared at 13 wks of age in DMSO-treatedmice (control), the WHI-P131—(50 mg/kg) treated mice started to developdiabetes at 22 wks of age (FIG. 27). At 25 wks, {fraction (22/37)} (60%)of control mice became diabetic, while only {fraction (2/11)} (18%) ofNOD females treated with 50 mg/kg of WHI-P131 developed diabetes (p=0.017). Daily treatment with 20 mg/kg of WHI-P131 did not show protectiveeffect on diabetes development—{fraction (5/9)} (56%) of treated micedeveloped diabetes till 18 wks of age (FIG. 27). We asked whether shortcourse of treatment with 100 mg/kg of WHI-P131, from wks 5 to 9 of life,could have a lasting protective effect. FIG. 28 shows that suchtreatment did not result with the protection from diabetesdevelopment—diabetes started at 10 wks and by 25 wks of age {fraction(10/12)} (83%) of NOD females became diabetic. Then, it was studiedwhether later beginning of treatment (at 10 wks of age) with 100 mg/kgof WHI-P131 could be effective in the prevention of diabetes. FIG. 28shows that treatment with 100 mg/kg of WHI-P131, initiated at 10 wks ofage, is as effective as treatment initiated earlier, at 5 wks ofage—diabetes incidence reached 9% ({fraction (1/11)}) at 25 wks of ageunder the first treatment (p=0.007 compared to controls) and 18%({fraction (4/22)}) under the later one (p=0.025 compared to controls).

[0228] Group of normoglycemic NOD females treated with DMSO (n=3) orwith 100 mg/kg of WHI-P131 for 20 (n=6) or 15 weeks (n=7) was fasted onthe end of experimental period (at 25 wks of age) and IPGTT wasperformed. Non-diabetic, non-treated C57BL/6 mice (n=5) were used ascontrols in IPGTT test, as well. The results—blood glucose levels weresimilar between the C57BL/6 mice and both groups of WHI-P131-treated NODmice during 120 min time period post glucose challenge. In contrast,normoglycemic DMSO-treated mice exhibited a significantly higher bloodglucose level at each observed time point then either ofWHI-P131-treated groups.

[0229] Further, histological examination of the insulitis level of thepancreata obtained from the mice analyzed in IPGTT was done. Insulitisscore of controls (n=3) was 1.43±0.15, while insulitis score of NODfemales treated by WHI-P131 from 5-25 wks of age was significantly (p=0.026) lower −0.86±0.12.However, insulitis score of NOD females treated byWHI-P131 from 10-25 wks of age was not different from controls(1.42±0.24).

[0230] Prevention of Diabetes Development in Adoptively TransferredNOD-scid/scid Females by WHI-P131 Treatment.

[0231] As WHI-P131 showed capability to prevent diabetes development inNOD females during the prediabetic phase of spontaneous development oftype I diabetes, next we wanted to determine whether WHI-P131 wascapable of suppressing effectors from already diabetic mice intransferring disease to NOD-scid mice. Two groups of NOD-scid femaleswere adoptively transferred with 1×10⁷ splenocytes from diabetic NODfemales and one group was treated daily from the day of transfer with 50mg/kg of WHI-P131 (n=12), while another one was treated by 10% DMSO(n=11) (FIG. 34). While {fraction (6/11)} (55%) of control NOD-scidbecame diabetic at 4 wk, followed by {fraction (8/11)} (73%) of diabeticmice at week 5 post adoptive transfer,-only {fraction (1/12)} (8%) ofWHI-P131-treated NOD-scid females became diabetic in the sameexperimental period (FIG. 4). Clearly, diabetes development postadoptive transfer was significantly (p<0.002) prevented by WHI-P131treatment.

[0232] The ability of a compound to prolong allograft survival can bedetermined using assays that are known in the art, or can be determinedusing assays similar to those described in Example 8.

Example 8 Prolongation of Allograft Survival Without Impairment of IsletCell Function

[0233] Male C57BL/6 mice (H-2^(b)) 8-12-wk-old were used as recipientsand BALB/c (H-2^(d)) males of the same age were used as a donors. Bothstrain of mice were purchased at Taconic, Germantown, N.Y., and housedunder pathogen-free conditions in Animal care facility of HughesInstitute. Jak3^(−/−) (C57BL/6×129/Sv, H-2^(b)) mice, homologous fordisrupted Jak3 gene were generous gift of Dr. J. N. Ihle, St. JudeChildren's Research Hospital, Memphis, Tenn. A homozygous Jak3^(+/+)mice were bred to C57BL/6 mice and the offspring of the Fl generationwere backcrossed to C57BL/6 mice. After three generations ofbackcrossing to C57BL/6, the offspring were intercrossed to produceJak3^(−/−) and wild-type (WT)Jak3^(+/+) mice. 10-12-wk-old Jak3^(−/−)and WT males were used as recipients of BALB/c islets.

[0234] Mixed Lymphocyte Reaction (MLR).

[0235] For MLR assay, responder cells (splenocytes obtained from10-wk-old C57BL/6 males) were plated in triplicates in 96-well-plates inthe concentration of 4×10⁵/100 μl of RPMI (Life Technologies , GrandIsland, N.Y.) with addition of 10% fetal bovine serum (LaboratoriesInc., Logan, Utah). Mitomycin-treated stimulators (splenocytes obtainedfrom 10-12-wk-old BALB/c males) were added in the concentration of 8×10⁴in 50 μl. WHI-P131 was added in different concentrations to a finalvolume of 200 μl and cells were cultured in 5% CO₂ with humidified airin an incubator at 37° C. for 5 days. Then, a calorimetric assay for thequantification of cell proliferation, based on the cleavage of thetetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells(Boehringer Mannheim, Indianapolis, Ind.), was performed followingmanufacturer's instructions. The absorbance was measured at 450/690 nmon a Multiskan MS microplate scanner. The value of cell proliferationwas obtained by diminishing O.D. value of proliferation of stimulatedcells by O.D. value of non-stimulated cells (O.D. values ofnon-stimulating cells were between 0.370-0.450).

[0236] Apoptosis Detection.

[0237] C57BL/6 splenocytes (3×10⁶/ml) were cultured in 24-well plate for20 h in 500 μl of RPMI-1640 under the conditions described above.WHI-P131 was added in the concentrations of 0.1, 1, 10 and 100 μg/ml.Apoptotic cell death was detected by TUNEL, using InSitu Cell DetectionKit, Fluorescein (Boehringer Mannheim, Indianapolis, Ind.). After theculture period, cells were washed, fixed, permeabilised and stainedfollowing manufacturer's instructions and apoptosis was analyzed by flowcytometry, using FACS Calibur (Becton Dickinson, San Jose, Calif.).

[0238] Flow Cytometric (FACS) Analysis.

[0239] Single cell suspension of splenocytes was prepared fromWHI-P131—(130 mg/kg/day) or vehicle-treated C57BL/6 mice, red bloodcells were lysed by lysis buffer and splenocytes (1×10⁶) were stainedwith 1:100 dilution of following anti-mouse monoclonal antibodies (Ab):anti-CD3-FITC Ab (clone 145-2C11), anti-CD4-FITC Ab (clone GK1.5),anti-CD8-PE Ab (clone 53-6.7) and anti-CD 19-PE Ab (clone 1D3). All Abswere purchased from Pharmingen, San Diego, Calif. Stained splenocyteswere analyzed by FACS Calibur, as described above.

[0240] Islet Isolation.

[0241] Islets of Langerhans were isolated from BALB/c males by the bileduct perfusion with 3-4 ml of collagenase P (3 mg/ml) (BoehringerMannheim, Indianapolis, Ind.) and deoxyribonuclease (0.1 mg/ml) (Sigma,St. Louis, Mo.), as described previously Cetkovic-Cvrlje M, et al.,1997, Diabetes, 46: 1975-1982). Islets were hand-picked 3-4 times underthe dissecting scope before islets were free of exocrine tissue,vessels, lymph nodes and ducts and ready for transplantation.

[0242] Allogeneic Islet Transplantation and Drug Treatment.

[0243] Four hundred islets were placed in Hamilton syringe andtransplanted under the left kidney capsule of each diabetic recipientmouse. Recipients were rendered diabetic with a single i.p. dose ofstreptozotocin (200 mg/kg; Sigma, St. Louis, Mo.) 1 wk beforetransplantation. Blood glucose level was measured by One Touch Profileglucose monitor system (Lifescan, Milpitas, Calif.). Only diabetic micewith glycemia over 350 mg/dl were used as transplant recipients.Allograft function was monitored by serial blood glucose measurements.Primary graft function was defined as a blood glucose under 200 mg/dl onday 3 post transplantation and graft rejection was defined as a rise inblood glucose exceeding 250 mg/dl on two consecutive measurements,following a period of primary graft function. Recipients were treateddaily with high-dose (20 mg/kg) of cyclosporine A (Sigma, St. Louis,Mo.), WHI-P131 (50 and 75 mg/kg, divided in three doses), WHI-P132 (50mg/kg, divided in three doses) or with vehicle control from the day oftransplantation until the day of rejection. All injections were giveni.p.

[0244] Intraperitoneal Glucose Tolerance Test (IPGIT) in Syngeneic IsletTransplant Recipients.

[0245] IPGTT was performed in C57BL/6 recipients that were transplantedwith syngeneic islet grafts (400 C57BL/6 islets) and treated withWHI-P131 or vehicle control for two months. Mice were fasted for 10hours, and the glucose solution (1.5 g/kg body weight) was injected i.p.Before and after injection of glucose, blood samples were taken andblood glucose levels were measured (as described above) at 0, 30, 60 and120 min time points.

[0246] Histopathological Studies.

[0247] The vehicle control—(n=3) and WHI-P131-treated C57BL/6 recipients(n=5) of islet allograft were sacrificed on day 14 post transplantation;Jak3 recipients (n=6) were sacrificed on day 100 post transplantation;the C57BL/6 recipients of syngeneic islets—vehicle control—(n=4) andWHI-P131-treated (n=5) were sacrificed on day 180 post transplantation.Kidneys bearing grafts were removed, fixed in 10% formalin and embeddedin paraffin. Serial sections of graft area were cut and stained withhematoxylin and eosin. For insulin staining, sections were stained byimmunoperoxidase method using 1:100 dilution of polyclonal guinea piganti-insulin antibody (Gpx Insulin, Dako, Carpinteria, Calif.) and 1:100dilution of secondary antibody conjugated to horse radish peroxidase(Guinea Pig Immunoglobulins HRP, Dako, Carpinteria, Calif.). Sectionswere briefly counterstained with hematoxylin and mounted for lightmicroscopic examination.

[0248] Statistical Analysis.

[0249] Statistical analysis was done by using unpaired Student's t-test(MLR and FACS data). Experimental differences in allograft rejectionsbetween the drug-treated and control groups were assessed by theKaplan-Meier life table analysis using Mantel-Cox test. The p value<0.05 was considered as statistically significant.

[0250] Results for Example 8

[0251] Jak3^(−/−) Mice do not Reject Islet Alograft.

[0252] JAK3-deficient males (n=6) and their WT littermates (n=7),rendered diabetic by STZ, were transplanted with BALB/c islets under thekidney capsule and blood glucose level was followed for 100 days posttransplantation. While islet allografts of WT controls were rejectedwith a MST of 12.9±1.1 days, all islet allografts of Jak3 recipientssurvived 100 days post transplantation (FIG. 35). Histological analysisof grafts showed no infiltration (data not shown) to slight mononuclearinfiltration of graft area with completely preserved islet morphology.

[0253] WHI-P131-induced Inhibition of MLR Response.

[0254] WHI-P131, added in the concentration of 0.1, 1, 10 and 50 μg/ml,inhibited proliferation of alloreactive splenocytes in MLR in adose-dependent manner (FIG. 36). While the concentration of 0.1·g/ml ofWHI-P131 induced statistically significant (p=0.0095) reduction of MLRresponse, complete abrogation of the response (obtained O.D. values werebelow the O.D. level of non-stimulating controls) was obtained withaddition of 1 μg/ml of WHI-P131 (FIG. 36). Next we tested whetherWHI-P131-induced lymphocyte death was the reason for inhibited MLRresponse. Therefore, apoptotic splenocytes (TUNEL-positive) weredetermined after the culture period of 20 h with addition of differentconcentrations of WHI-P131. FIG. 37 shows that apoptotic cell death ofsplenocytes cultured with addition of 0.1 and 1 μg/ml of WHI-P131 doesnot differ from control cells, while addition of 10 and 100 μg/mlsignificantly increased (p=0.008 and p<0.0001, respectively) apoptoticcell death in comparison to control cell death during the observedculture period. Therefore, WHI-P131 effects on MLR suppression, obtainedwith lower concentration of the drug (0.1 and 1 μg/ml), seems to not becaused by the induction of cell death.

[0255] WHI-P131 Treatment of Recipients Prolonged Islet AllograftSurvival.

[0256] The control C57BL/6 mice (n=39) rejected BALB/c islet allograftwith a mean survival time (MST) of 14.1±0.9 days. Daily treatment ofrecipients (n=10) with high-dose—20 mg/kg—of CsA (18) significantlyincreased (p=0.0005) allograft survival (MST=27.9±4.6 days) (FIG. 38).Treatments with 50 mg/kg (n=14) or 75 mg/kg (n=14) of WHI-P131 were aseffective as CsA treatment in prolongation of allograft survival(MST=24.7±3.4 and 25.3±3.8, respectively) in comparison to controls(p=0.0002 and 0.001, respectively) (FIG. 38).

[0257] Histologic examination of the islet allografts that wereharvested at 14 days post transplantation from normoglycemic recipientstreated with vehicle control (n=3) showed lymphocytic invasion andmassive islet destruction, clearly seen with immunostaining for insulin.This finding is in striking contrast with allografts from recipientstreated with 50 mg/kg of WHI-P131 (n=5), in which lymphocyticinfiltration was present but without extensive destruction of islets.The hyperglycemic vehicle-treated allografts (n=4), harvested at thesame time point, were completely invaded by lymphocytes with noremaining insulin-producing cells.

[0258] The next experiment was performed with an aim to study theeffects of WHI-P131 treatment on splenocyte populations in vivo. C57BL/6males were treated with 130 mg/kg of WHI-P131 (n=7) and with vehiclecontrol (n=5) for 10 days. Total number of splenocytes was 154±8.5×10⁶in vehicle-treated controls, 6 while reduced number ofsplenocytes—119±9.4×10⁶ was found in WHI-P131-treated mice (p=0.025).There were no differences in the percentages of CD3+, CD4+ and CD8+T-cells and CD19+ B-cells between the WHI-P131- and vehicle-treated mice(data not shown). However, FIG. 6 shows that slight differences in thenumber of studied cell populations between the WHI-P131-treated andcontrol groups were found. Thus, the number of CD3+ splenocytes wasreduced to 37.7±3.2×10⁶ (however, not statistically significant,p=0.0617) in WHI-P131-treated mice in comparison to 46.9±2.6×10⁶ incontrols. The number of CD4+ T-cells -16.8±1.2×10⁶ (p=0.0416), as wellas B-cells -57.1±5.3×10⁶ (p=0.0267) were reduced in WHI-P131-treatedmice compared to the controls -20.5±0.6×10⁶ and 78.2±6.1×10⁶,respectively (FIG. 39).

[0259] Function of Transplanted Syngeneic Islets is Preserved after theLong-term Treatment with WHI-P131.

[0260] This study was performed to test the effect of long-termtreatment (180 days) with 50 mg/kg of WHI-P131 on syngeneic islet graftfunction. As it could be seen on FIG. 40, non-fasting blood glucoselevel did not differ during the entire experimental period of 180 dayspost transplantation between the vehicle control—(n=4) andWHI-P131-treated (n=5) syngeneic recipients. IPGTT was performed on day70 of treatment. The nonfasting blood glucose of the controls andP131-treated recipients at that time was 102.0±6.7 and 124.6±10.8 mg/dl,respectively. IPGTT test showed that there was no significant differencein islet function of non-transplanted, non-treated C57BL/6 males,transplanted vehicle-treated recipients (controls) and transplantedWHI-P131-treated recipients (FIG. 41). Another IPGTT test was performedon the end of experimental period (day 180 post transplantation). Thenon-fasting blood glucose level at that time was 120.9±12.5 in vehiclecontrol- and 115.2±9.2 mg/dl in WHI-P131-treated recipients. IPGTT testshowed again that islet function of WHI-P131-treated recipients was notdifferent from islet function of either control recipients ornon-transplanted C57BL/6 mice.

[0261] Representative examples from the light microscopical evaluationof the vehicle control- and WHI-P131-treated syngeneic islet grafts areillustrated in FIG. 8. Hematoxylin and eosin staining of vehicle controland WHI-P131-treated grafts showed that there were no significantmorphological changes between them. Immunohistochemical analysis.confirmed that insulin expression in WHI-P131-treated grafts wascomparable to that of the grafts of the vehicle control-treatedrecipients.

[0262] All publications, patents, and patent documents are incorporatedby reference herein, as though individually incorporated by reference.The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A compound of formula I:

wherein: X is HN, R₁₁N, S, O, CH₂, or R₁₁CH; R₁₁ is hydrogen,(C₁-C₄)alkyl, or (C₁-C₄)alkanoyl; R₁-R₈ are each independently hydrogen,hydroxy, mercapto, amino, nitro, (C₁-C₄)alkyl, (C₁-C₄)alkoxy,(C₁-C₄)alkylthio, or halo; wherein two adjacent groups of R₁-R₅ togetherwith the phenyl ring to which they are attached may optionally form afused ring, for example forming a naphthyl or a tetrahydronaphthyl ring;and further wherein the ring formed by the two adjacent groups of R₁-R₅may optionally be substituted by 1, 2, 3, or 4 hydroxy, mercapto, amino,nitro, (C₁-C₄)alkyl, (C₁-C₄)alkoxy, (C₁-C₄)alkylthio, or halo; and R₉and R₁₀ are each independently hydrogen, (C₁-C₄)alkyl, (C₁-C₄)alkoxy,halo, or (C₁-C₄)alkanoyl; or R₉ and R₁₀ together are methylenedioxy; ora pharmaceutically acceptable salt thereof; provided the compound is not4-(4′-hydroxyl-phenyl)-amino-6,7-dimethoxyquinazoline.
 2. The compoundof claim 1 wherein X is R₁₁N.
 3. The compound of claim 1 wherein X isHN.
 4. The compound of claim 1 wherein each of R₁, R₂, R₄, R₅, R₆, R₇,and R₁₀ is H.
 5. The compound of claim 1 wherein R₃ is (C₁-C₄)alkoxy,hydroxy, nitro, halo, trifluoromethyl, or NR₁₂R₁₃ wherein R₁₂ and R₁₃are each independently hydrogen, (C₁-C₄)alkyl, (C₁-C₄)alkenyl,(C₁-C₄)alkynyl, (C₃-C₈)cycloalkyl, or heterocycle.
 6. The compound ofclaim 1 wherein R₃ is hydroxy.
 7. The compound of claim 1 wherein R₂ orR₃ is hydroxy.
 8. The compound of claim 1 wherein R₂ or R₃ is hydroxy;and one of R₁-R₅ is halo.
 9. The compound of claim 1 wherein R₂ or R₃ ishydroxy. 10 The compound of claim 1 wherein is (C₁-C₄)alkoxy.
 11. Thecompound of claim 1 wherein R₉ is (C₁-C₄)alkoxy.
 12. The compound ofclaim 1 which is 4-(3′-hydroxyl-phenyl)-amino-6,7-dimethoxyquinazoline;4-(3′,5′-dibromo-4′-hydroxyl-phenyl)-amino-6,7-dimethoxyquinazoline or4-(3′-bromo-4′-hydroxyl-phenyl)-amino-6,7-dimethoxyquinazoline; or apharmaceutically acceptable salt thereof.
 13. A pharmaceuticalcomposition comprising a compound of formula I:

wherein: X is HN, R₁₁N, S, O, CH₂, or R₁₁CH; R₁₁ is hydrogen,(C₁-C₄)alkyl, or (C₁-C₄)alkanoyl; R₁-R₈ are each independently hydrogen,hydroxy, mercapto, amino, nitro, (C₁-C₄)alkyl, (C₁-C₄)alkoxy,(C₁-C₄)alkylthio, or halo; wherein two adjacent groups of R₁-R₅ togetherwith the phenyl ring to which they are attached may optionally form afused ring, for example forming a naphthyl or a tetrahydronaphthyl ring;and further wherein the ring formed by the two adjacent groups of R₁-R₅may optionally be substituted by 1, 2, 3, or 4 hydroxy, mercapto, amino,nitro, (C₁-C₄)alkyl, (C₁-C₄)alkoxy, (C₁-C₄)alkylthio, or halo; and R₉and R₁₀ are each independently hydrogen, (C₁-C₄)alkyl, (C₁-C₄)alkoxy,halo, or (C₁-C₄)alkanoyl; or R₉ and R₁₀ together are methylenedioxy; ora pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier.
 14. The composition of claim 13 wherein R₂ or R₃ ishydroxy.
 15. The composition of claim 13 wherein R₂ or R₃ is hydroxy;and one of R₁-R₅ is halo.
 16. The composition of claim 13 wherein thecompound of formula I is4-(3′-hydroxyl-phenyl)-amino-6,7-dimethoxyquinazoline;4-(3′,5′-dibromo-4′-hydroxyl-phenyl)-amino-6,7-dimethoxyquinazoline or4-(3′-bromo-4′-hydroxyl-phenyl)-amino-6,7-dimethoxyquinazoline; or apharmaceutically acceptable salt thereof.
 17. A pharmaceuticalcomposition comprising4-(4′-hydroxyl-phenyl)-amino-6,7-dimethoxyquinazoline; or apharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier.
 18. A therapeutic method for treating leukemia orlymphoma in a mammal comprising administering to the mammal in needthereof an effective amount of a JAK-3 inhibitor.
 19. A therapeuticmethod for treating or preventing organ transplant rejection in a mammalcomprising administering to the mammal in need thereof an effectiveamount of a JAK-3 inhibitor.
 20. A therapeutic method for preventing orreducing ultraviolet B radiation-induced inflammatory response in amammal comprising administering to the mammal in need thereof aneffective amount of a JAK-3 inhibitor.
 21. A therapeutic method forinhibiting the release of prostaglandin E₂ in a mammal comprisingadministering to the mammal in need thereof an effective amount of aJAK-3 inhibitor.
 22. A therapeutic method for preventing or reducingUVB-induced skin edema or vascular permeability changes in a mammalcomprising administering to the mammal in need thereof an effectiveamount of a JAK-3 inhibitor.
 23. A therapeutic method for preventing orreducing ultraviolet B radiation-induced damage to epithelial cells ormutation frequency in skin in a mammal comprising administering to themammal in need thereof an effective amount of a JAK-3 inhibitor.
 24. Atherapeutic method for protecting a mammal from tumorigenic effects ofUVB light comprising administering to the mammal in need thereof aneffective amount of a JAK-3 inhibitor.
 25. A therapeutic method forinhibiting T-cell activity in a mammal comprising administering to themammal in need thereof an effective amount of a JAK-3 inhibitor.
 26. Atherapeutic method for preventing or treating an autoimmune diseasecomprising administering to the mammal in need thereof an effectiveamount of a JAK-3 inhibitor.
 27. A therapeutic method for preventing ortreating graft-verses host disease comprising administering to themammal in need thereof an effective amount of a JAK-3 inhibitor.
 28. Themethod of any one of claims 18-27 wherein the compound is a compound ofclaim
 1. 29. The method of any one of claims 18-27 wherein the JAK-3inhibitor is 4-(4′-hydroxyl-phenyl)-amino-6,7-dimethoxyquinazoline; or apharmaceutically acceptable salt thereof.